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Molecular and Cellular Biology, January 2000, p. 478-487, Vol. 20, No. 2
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
CLN1 and Its Repression by Xbp1 Are
Important for Efficient Sporulation in Budding Yeast
Bernard
Mai and
Linda
Breeden*
Fred Hutchinson Cancer Research Center,
Division of Basic Sciences, Seattle, Washington 98109-1024
Received 9 July 1999/Returned for modification 27 September
1999/Accepted 13 October 1999
 |
ABSTRACT |
Xbp1, a transcriptional repressor of Saccharomyces
cerevisiae with homology to Swi4 and Mbp1, is induced by stress
and starvation during the mitotic cycle. It is also induced late in the
meiotic cycle. Using RNA differential display, we find that genes
encoding three cyclins (CLN1, CLN3, and
CLB2), CYS3, and SMF2 are
downregulated when Xbp1 is overexpressed and that Xbp1 can bind to
sequences in their promoters. During meiosis, XBP1 is
highly induced and its mRNA appears at the same time as
DIT1 mRNA, but its expression remains high for up to
24 h. As such, it represents a new class of meiosis-specific
genes. Xbp1-deficient cells are capable of forming viable gametes,
although ascus formation is delayed by several hours. Furthermore, Xbp1
target genes are normally repressed late in meiosis, and loss of
XBP1 results in their derepression. Interestingly, we find
that a deletion of CLN1 also reduces the efficiency of
sporulation and delays the meiotic program but that sporulation in a
cln1 xbp1 strain is not further delayed.
Thus, CLN1 may be Xbp1's primary target in meiotic cells.
We hypothesize that CLN1 plays a role early in the meiotic
program but must be repressed, by Xbp1, at later stages to promote
efficient sporulation.
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INTRODUCTION |
The absence of a fermentable carbon
source combined with the limitation for nitrogen elicits a
developmental switch in the yeast Saccharomyces cerevisiae
from mitotic growth to meiosis and gametogenesis. The progress through
this pathway, which ultimately results in the formation of an ascus
containing four haploid spores, is thought to be regulated mainly at
the level of transcription. Based on their temporal sequence of
expression, meiotic genes can be divided into at least four classes:
early, middle, mid-late, and late (reviewed in reference
24). Entry into the meiotic program is triggered by
nutritional and mating type signals which lead to the activation of the
transcription factor Ime1 (21, 44). In response to
phosphorylation by the Rim11 kinase, the activation domain of Ime1
binds to the DNA-binding protein Ume6 and converts it from a repressor
to an activator (40). This complex induces the transcription
of early genes which have URS1 elements in their promoters (5,
6). Among these early genes is Ime2, a protein kinase homolog
involved in downregulating Ime1 and necessary for the expression of
other early and middle genes (19, 31, 43). The gene for the
DNA-binding protein Ndt80 is transcribed in the middle of the
transcriptional program of meiosis and activates its own expression as
well as that of other middle and mid-late genes which are required for
meiotic division (e.g., genes encoding B-type cyclins [Clbs]) and
spore formation (e.g., SPS1) (11). Ndt80 was
shown to bind in vitro to MSEs (mid-sporulation elements) which are
found in many meiotically induced genes (11). The regulatory
cascade which activates mid-late and late genes is undefined. There is
evidence that the protein kinases Sps1, a Ste20 homolog, and Smk1, a
mitogen-activated protein kinase homolog, stimulate these late genes
(17, 23). It is also possible that the completion of meiotic
DNA synthesis generates a signal required for the transcription of
later sporulation-specific genes (30).
The cyclin-dependent kinase Cdc28 is essential for progress through
mitosis and meiosis (33, 42). During the mitotic cell cycle
there are four bursts of cyclin transcription, and each produces
cyclins with distinct roles in mitotic progression. The first wave
produces Cln3, which is key for inducing the second wave of cyclins,
Cln1,2 and Clb5,6. These pairs of cyclins play redundant roles in
inducing budding and DNA replication. Then Clb3,4 and later Clb1,2 are
made. These, too, have overlapping functions in spindle assembly and
mitosis, with Clb2 having a predominant role (for a review see
reference 27). All Clbs are also expressed during
meiosis (11, 18). The meiotic expression of the six Clbs,
with the exception of Clb2, is controlled by the meiosis-specific
transcription factor Ndt80, which is expressed during the same time
interval (11). Clb1, Clb4, Clb5, and Clb6 have been shown to
be the most important meiotic cyclins, and the absence of one or
several of them causes a drastic drop in sporulation efficiency
(13, 15, 18, 45). In contrast, Clb2, which is of primary
importance in mitosis, plays a lesser role in meiosis (13,
18).
Recent experiments have suggested that the G1 cyclins play
a negative role in sporulation (12), but this has not been
thoroughly investigated. The absence of any one of three
G1-specific cyclins Cln1, -2, and -3 does not eliminate
spore formation (15), and deletion of CLN3
appears to speed up the meiotic program (12). In addition,
overproduction of G1 cyclins inhibits sporulation, probably
by transcriptional repression of Ime1, a key regulator for entering
meiosis (12). Interestingly, the combination of CLN3 downregulation and ectopic expression of
IME1 is sufficient to cause cells to enter meiosis in rich
media (12).
The XBP1 gene of S. cerevisiae is of interest
because it shares homology to the DNA-binding domain of Swi4 and Mbp1
and binds a related sequence (29). XBP1 mRNA is
induced by a variety of stress conditions, including heat shock, high
osmolarity, oxidative stress, DNA damage, and glucose starvation.
Interestingly, Xbp1 expression is dramatically downregulated when
wild-type cells evolve for 250 generations under glucose-limiting
conditions (16). Consistent with this observation,
overexpression of XBP1 causes slow growth and repression of
the G1 cyclins Cln1, Cln2, and Cln3 (29). When
Xbp1 is fused to the LexA DNA-binding domain, it acts as a
transcriptional repressor (29). In order to use a more
global approach to identify targets of this putative repressor, we used
the method of RNA differential display and monitored the effect of loss
of Xbp1 on the meiotic cycle.
In this report, we describe the identification of CLN1,
CLN3, CLB2, CYS3, and SMF2
as target genes of Xbp1. All of these genes have Xbp1 binding sites in
their promoters, and evidence is provided that Xbp1 binds these
sequences in vitro. Furthermore, we show that XBP1 is
expressed late in meiosis and that it represents a new class of
meiosis-specific genes whose transcript level increases after meiotic
DNA replication is completed and is maintained at a high level
throughout gametogenesis. In xbp1 cells, ascus formation is delayed and Xbp1 target genes are derepressed late in meiosis. The
most dramatic derepression is observed for CLN1. To see if expression of CLN1 late in gametogenesis could be
responsible for the delay of spore formation observed in
xbp1 mutants, we deleted CLN1 in
XBP1 and xbp1 cells. Deletion of
CLN1 alone leads to a delay of spore formation, but deletion
of XBP1 leads to no further delay. This finding suggests
that CLN1 plays a role early in gametogenesis, but its
presence is deleterious later in the process. These findings identify
Xbp1 as a novel transcriptional repressor in yeast which facilitates
gametogenesis, perhaps by repressing key mitotic cyclins and other genes.
| |
MATERIALS AND METHODS |
Yeast strains, growth conditions, and cell analysis.
BY2059
(xbp1::HIS3) transformed with BD2014
(pGAL:XBP1) (29) was used for the differential
display screen. Cells were grown at 30°C unless otherwise specified
in either YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) or
synthetic complete medium supplemented with amino acids as appropriate
to select for transformants (2). G418 (200 µg/ml; Gibco
BRL) was added to YEPD to select for the presence of the kanamycin
resistance gene (kanR); 2% galactose was added
to a culture grown in 2% raffinose to induce expression from the
GAL1 promoter, and 2% glucose was added to repress
expression. Transformation and tetrad analysis were performed as
previously described (2).
The yeast strains used in this study are listed in Table
1. SK-1 derivative strains BY1633 (=NKY
278), BY1593, BY1594, BY2386, BY2387, BY2388, BY2673, BY2674, BY2675,
BY2676, BY2677, and BY2678 were used to synchronously induce meiosis
(1, 20). To determine the role of Xbp1 in the selection of
double-strand break (DSB) sites SK-1 derivative strains BY2533 and
BY2534 were used. All disruptions of XBP1 were confirmed by
PCR using oligonucleotide primers described in reference
29. To sporulate yeasts in liquid medium, cultures
were grown in YEPD to an optical density (OD) of 2 to 3, diluted to an
OD of 0.1 into YEPA (1% yeast extract, 2% peptone, 2% potassium
acetate), and grown overnight with vigorous shaking to an OD of 1 to
1.5. Cells were harvested by centrifugation, washed once with water,
resuspended to the same cell density in sporulation medium (1%
potassium acetate), and incubated at 30°C with vigorous shaking. At
indicated times, aliquots were harvested by centrifugation, and the
pellets were stored at 
80°C. The time of transfer to sporulation
medium is referred to as 0 h.
DNA content was quantitated by fluorescence-activated cell sorting
(FACS) analysis using a Becton Dickinson FACScan and CellQuest
software
as described previously (
29). Spore formation was scored
by
light microscopy (640× magnification; Zeiss, Jena, Germany).
The
average of at least four fields was
calculated.
Detection of DSBs.
Diploid cells homozygous for the
rad50S allele do not process or repair the ends of DSBs, and
therefore the DSB fragments accumulate as discrete species
(1). Cells (200 ml) from rad50S strains carrying
either a wild-type or a deleted allele of XBP1 were
sporulated, and at each time point 25 ml of cells was removed and mixed
with 25 ml of ice-cold 100% ethanol. Chromosomal DNA was prepared as
described previously (39) and digested with HindIII. The fragments were separated on a 0.7% agarose
gel and blotted to GeneScreen (Dupont, NEN), and hybridizations were
done as described elsewhere (29). The probe was a 1,970-bp
EcoRI fragment from the 3' end of the CYS3 gene
labeled by random priming with [
-32P]dCTP.
DNA manipulations.
For the expression of full-length Xbp1 in
Escherichia coli, an NdeI-XhoI
fragment from BD2013 (29) was cloned into pET14b (Novagen)
to generate a His6 N-terminal fusion to Xbp1 (BD2207). To
create His6-Xbp11-509, BD2207 was cut with
SalI, digested for a limited time with Bal 31 nuclease, recut with NdeI, and ligated back into pET14b,
generating BD2208.
For construction of the
XBP1-kanR disruption
cassette, long flanking homology PCR (
47) was used. In the
first PCR, two separate
fragments, corresponding to amino acids 1 to 98 in Xbp1 and 577
to 647 (including 122 nucleotides downstream of
XBP1), were generated,
using 50 ng genomic DNA of BY2058
(
29) as the template; 300
ng of each product from the first
PCR together with 1 µM outermost
5' and 3' primers (BL152 and BL153)
(
29) were used to synthesize
the 2.1-kb
XBP1-kanR disruption cassette from 20 ng of
template (pFA-kanMX6 [
47]).
The PCR fragments were
purified through agarose gels, and 100
to 300 ng of fragment was used
for each yeast
transformation.
Differential display analysis.
Total RNA was prepared from
BY2059 that was transformed with the empty expression vector or with
pGAL:XBP1, grown in 2% raffinose, and shifted for 80 min to
2% galactose. To remove chromosomal DNA from the RNA preparation, 50 µg of total RNA was incubated with 10 U of RNase inhibitor and 10 U
of RNase-free DNase I (Promega) in 10 mM Tris-HCl (pH 8.3)-100 mM
KCl-1.5 mM MgCl2 in a total volume of 50 µl. After 30 min at 37°C, the RNA was extracted once with phenol, precipitated
with ethanol, and dissolved in 20 µl of H2O. Differential
display of Xbp1 overexpression compared to vector control RNA was
carried out as described by Liang and Pardee (28).
In a total volume of 20 µl, 200 ng of each RNA was reverse
transcribed by using 200 U of SuperScript II reverse transcriptase
(Gibco BRL) in 0.01 mM dithiothreitol-20 µM deoxynucleoside
triphosphates
(dNTPs)-1 µM oligo(dT) anchored primer
(T
12MA, T
12MC, T
12MG, or
T
12MT, where M can be A, C, or G). After 50 min of
incubation
at 37°C, the reverse transcriptase was inactivated by
heating
the reaction mixture for 5 min to 95°C. Four different cDNA
syntheses
were performed per RNA, each using a different
T
12MN oligo(dT)
primer. All of the following PCRs were done
in duplicate to exclude
PCR artifacts. Two microliters of each cDNA
reaction mixture was
PCR amplified with the same T
12MN
oligo(dT) primer (1 µM) and
an arbitrary decamer primer (0.2 µM) in
a total volume of 20 µl
containing 1 U of
Taq polymerase
(Fisher), 2 µM dNTPs, and 1 µCi
of [
-
33P]dCTP
(Dupont, NEN). The arbitrary primers (kit OPA-G) were purchased
from
Operon Technologies Inc. (Alameda, Calif.); the individual
sequences
can be retrieved online
(
http://web712d0.ntx.net/ss2b1/stockproducts/kkitd.html).
PCR
amplification was carried out in 40 cycles as follows: 94°C
for
30 s, 40°C for 2 min, and 72°C for 30 s, followed by a
5-min
extension at 72°C. Prior to loading onto a 6% denaturing
polyacrylamide
gel, a 3.5-µl aliquot of each reaction was mixed with
2 µl of
formamide loading buffer and heat denatured for 3 min at
95°C.
The gel was dried onto Whatman 3MM paper without fixation and
autoradiographed overnight. The bands of interest were cut out
of the
gel and soaked in 100 µl of H
2O for 10 min at room
temperature
and 15 min at 100°C. The cDNA in the supernatant was
recovered
by ethanol precipitation, using 50 µg of glycogen
(Boehringer
Mannheim) as the carrier. The cDNA was dissolved in 10 µl
of H
2O,
from which 4 µl was used for reamplifications in
a total volume
of 20 µl, using the same primer combination and PCR
conditions
except that the dNTP concentration was 20 µM and the
isotope was
omitted. Most cDNA fragments had to be reamplified twice
with
1 µl of the first reamplification as the template. The amplified
DNA was purified through an 1.5% agarose gel. The isolated DNAs
were
used directly as probes for Northern blots or as templates
for cycle
sequencing using the appropriate decamer primer and
around one-fifth of
the isolated DNA. For nearly all cDNAs, unambiguous
sequences were
obtained.
A total of 20 decamer primers were used. Whereas most of the PCR bands
were reproducible, only 50 to 60% showed the expected
regulation
pattern on Northern blots. In general, only fragments
between 100 and
500 nucleotides in length were
considered.
Gel retardation assay.
Gel retardation assays were performed
as described previously (29), using full-length
His6-Xbp1. The double-stranded 32P-labeled
oligonucleotide probes were the consensus binding site oligonucleotide,
a mutant consensus sequence to which Xbp1 does not bind
(29), CY3 (5'-TCGACATAAAAATCCTCGAGGAAAAGAA-3'),
and CL3 (5'-TCGATCTGTACTTTCCTCGAGCTTTTAATCTTCTT-3')
(only the upper strands are shown). Competition experiments
included a 500-fold molar excess of unlabeled competitor DNA over
labeled probe.
RNA analyses.
Total yeast RNA was analyzed by Northern
blotting as previously described (29). Hybridizations were
done simultaneously or sequentially to the following random-labeled
probes, which were generated by PCR using yeast
GENEPAIRS primer (Research Genetics, Huntsville, Ala.) and yeast genomic DNA: CLB2 (YPR119w),
CYS3 (YAL012w), DIT1 (YDR403w), DMC1
(YER179w), NDT80 (YHR124w), SGA1 (YIL099w),
SMF2 (YHR050w), SPS100 (YHR139c), and
TCM1 (YOR063w). Probes for XBP1, ACT1,
CLN1, and CLN3 were generated as described in
reference 29.
Antibodies.
To generate polyclonal antibodies directed
against Xbp1, His6-tagged full-length Xbp1 or a
His6-tagged C-terminal deletion of Xbp1
(His6-Xbp11-509) was expressed in E. coli BL21(DE3)pLysS (Novagen) and purified by nickel chelate
chromatography essentially as described previously (29). To
further purify Xbp1, the eluate was diluted 1:1 with binding buffer (20 mM Tris [pH 7.5], 100 mM NaCl, 1 mM MgCl2, 2% glycerol,
0.1% NP-40) and loaded on a heparin-Sepharose CL-6B column
(Pharmacia). The column was washed with 200 mM NaCl in binding buffer,
and bound proteins were eluted with a 200 to 1,000 mM NaCl gradient in
binding buffer. Xbp1 eluted at around 500 mM NaCl. These preparations
were used to inject rabbits (500 µg per injection) five times with
incomplete Freund's adjuvant. Antibodies were stored in aliquots at
80°C.
Immunoprecipitations and Western blot analysis.
Protein
extracts were prepared by glass bead disruption as described elsewhere
(2), using radioimmunoprecipitation assay buffer (150 mM
NaCl, 50 mM Tris [pH 7.5], 2 mM EDTA, 0.1% sodium dodecyl sulfate
[SDS], 1 Triton X-100, 50 mM NaF, 1 µg of leupeptin per ml, 1 µg
of pepstatin per ml, 1 mM phenylmethylsulfonyl fluoride). For
immunoprecipitations, 400 µg of protein extract was precleared with
preimmune serum coupled to protein A-Sepharose beads for 1 h at
4°C. The supernatant was recovered and incubated with 2.5 µl of
immune serum coupled to protein A-Sepharose beads for 4 h at
4°C. The beads were washed four times with radioimmunoprecipitation buffer; bound proteins were eluted with SDS sample buffer, boiled, and
loaded onto a 6.5% SDS gel. Following electrophoresis, proteins were
transferred to nitrocellulose (Micron Separations Inc.), and Western
blot analyses were performed, using a polyclonal antibody against Xbp1
at a dilution of 1:5,000 and a protein A-horseradish peroxidase
conjugate (Bio-Rad Laboratories) at a dilution of 1:3,000. Enhanced
chemiluminescence (Dupont, NEN) was used for detection.
| |
RESULTS |
Differential display of mRNAs from Xbp1-overexpressing cells.
The aim of this study was to understand the function of Xbp1. Xbp1 was
tentatively classified as a transcriptional repressor because (i) when
it is fused to the LexA DNA-binding domain it can repress the
expression of a reporter gene and (ii) overexpression of Xbp1 results
in the reduction of transcript levels of genes like CLN1,
which has Xbp1 binding sites in its promoter. In addition, high levels
of Xbp1 are induced under conditions of stress, and this is correlated
with reduced transcript levels of CLN1 and other putative
target genes. However, elimination of Xbp1 does not lead to
derepression of CLN1 during stress (29). Thus, if Xbp1 is a repressor of CLN1 transcription, it cannot be the
only one. In hopes of clarifying the role of Xbp1, we used the method of differential display to identify in vivo target genes.
In our first attempt to identify direct or indirect targets of the
DNA-binding protein Xbp1, we compared the pattern of genes
transcribed
in wild-type and Xbp1-deficient cells under stress
using the
differential display method developed by Liang and Pardee
(
28). With the number of primer combinations that we used,
it
should have been possible to detect nearly every expressed gene
(
3). Nevertheless, we were not able to identify any
reproducible
differences (data not shown), which suggests that
Xbp1-mediated
repression has no role in the stress response of yeast or
that
there are other factors with similar activity. A recent study
makes the latter explanation more likely. Mitotic cells were grown
continuously for 250 generations under glucose limitation and
then
subjected to microarray analysis. Xbp1 was one of the 39
known genes
that the cells evolved to repress under these conditions
(
16).
For our second attempt, we induced high-level expression of Xbp1 in
xbp1 cells. Four different oligo(dT) primer pools were
used to reverse transcribe total RNA isolated from cells overexpressing
Xbp1 under the control of the
GAL1 promoter on pYES2 and
from
control cells treated equivalently but harboring the pYES2 vector
only (see Materials and Methods). The cDNAs were PCR amplified
in
the presence of [
-
33P]dCTP, using 20 different
arbitrary decamer primers, and the
products were analyzed on denaturing
polyacrylamide gels. In this
way, 80 primer combinations were used to
screen for genes which
show changes in expression when Xbp1 is
overexpressed.
RNA was collected from the Xbp1-overexpressing cells that had been
grown in 2% raffinose and shifted for 80 min to 2% galactose.
Although the maximum level of
XBP1 mRNA is reached after 30 min
(
29), a later time point was chosen to allow for the
Xbp1 protein
to have an effect on potential target genes. This was done
because
we have observed, in stressed cells and during meiosis, a
20-min
to 1-h lag between induction of
XBP1 mRNA and
appearance of Xbp1
protein (data not shown and Fig.
5). Using 80 different primer
combinations, we were able to detect four reproducible
instances
where Xbp1 overproduction reduced gene expression, examples
of
which are shown in Fig.
1. This number
of primer combinations
should have enabled us to observe expression
differences for nearly
all yeast genes (
3). Since only four
reproducible differences
were found, we conclude that Xbp1
overexpression does not have
global effects on transcription. Rather,
Xbp1 appears to affect
specific target genes, and in each case it acts
to repress transcription.
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FIG. 1.
Identification of Xbp1-regulated genes by differential
display. (A) Differential display using total RNA from control cells
(pGAL) and cells overexpressing Xbp1 (pGAL:XBP1).
Both strains are xbp1. PCRs were done in duplicate, and
the products were separated on 6% denaturing polyacrylamide gels. The
following primer combinations were used: OPA-G2-T12MC for
CLB2, OPA-G15-T12MC for CLN3, and
OPA-G16-T12MA for CYS3 and XBP1.
Arrows indicate RNAs that are differentially expressed and were
analyzed further. The identity of the corresponding gene was obtained
by sequencing (see Materials and Methods). (B) Northern blot analysis
using RNA preparations of the same strains grown in raffinose (Raf),
shifted for 80 min to glucose (Glu), or shifted for 30 or 80 min from
raffinose to galactose (Gal). Identical blots were hybridized to the
indicated probes. The ribosomal protein L3 gene TCM1 served
as a loading control. The bottom panel shows the ethidium bromide
(EtBr)-stained gel before it was blotted.
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Identification of genes downregulated by Xbp1.
To identify the
genes corresponding to the differentially regulated messages, the cDNA
fragments were isolated from the gel and twice reamplified with the
same set of primers. These PCR-amplified products were directly cycle
sequenced by using the upstream decamer primer (8).
Sequences of the cDNA fragments were compared to those in the
Saccharomyces genome database maintained at Stanford University. Downregulated genes were identified as CLB2,
CLN3, CYS3, and SMF2, with
CYS3 being amplified with several different primer
combinations. Clb2 is a G2-specific B-type cyclin
(46), Cln3 is a G1 cyclin (38), and
Cys3 is a cystathionine gamma-lyase which catalyzes the biosynthesis of
cysteine (35). The function of Smf2 is unknown. It was
cloned as a high-copy-number suppressor of a lethal mutation in the
yeast mitochondrial processing-enhancing protein (48). With
the exception of XBP1 in the control sample, none of the
differentially regulated bands disappeared completely, suggesting that
Xbp1 downregulates these genes but does not eliminate their expression
completely. The expression of very few genes was upregulated. Among
those, XBP1 itself was isolated with three different primer
combinations, as expected.
To confirm Xbp1 regulation of transcripts identified and isolated by
differential display, Northern blot analysis was performed
with probes
comprising the entire coding regions of candidate
genes. As shown in
Fig.
1B, all identified genes showed a downregulation
that is dependent
on the overexpression of Xbp1. The G
1 cyclin
gene
CLN1 was included in this analysis as a gene known to be
repressed under these conditions from our previous work
(
29).
Consistent with the differential display data (Fig.
1A), none
of the genes are completely shut off, but they are three- to
fourfold
downregulated.
Candidate target genes have Xbp1 binding sites in their
promoters.
To see if Xbp1 could be directly affecting these target
genes, we screened 1,000 bp upstream of the ATG of each candidate gene
for the existence of potential Xbp1 binding sites. Indeed, in each of
these promoter sequences, at least one sequence closely resembling the
Xbp1 consensus binding site was found (29). As shown in Fig.
2, the CLN1, CLB2,
CYS3, and SMF2 promoters have several potential
Xbp1 binding sites, and all of the potential binding sites noted share
the 100% conserved core sequence TCGA. The two CLN1 sites
are only 17 bp apart and were the first Xbp1 binding sites to be
characterized (29).
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FIG. 2.
Comparison of potential Xbp1 binding sites in the
promoters of candidate Xbp1 target genes to the Xbp1 consensus binding
site. The sequence 1,000 bp upstream the ATG of each gene was screened
for Xbp1 binding sites containing at least the four core bases (TCGA).
Nucleotide coordinates refer to the 5'-most residue of each sequence.
Black boxes mark positions identical to the site selection consensus
binding site. Asterisks mark binding sites used in in vitro binding
experiments.
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|
We also tested the ability of recombinant Xbp1 to bind to two of these
sites in gel retardation assays. The binding of Xbp1
to the consensus
binding site [GcCTCGA(G/A)G(C/A)g(a/g)] (
29)
was compared to its ability to bind the potential binding site
in the
CLN3 promoter (position
408 relative to the ATG) and the
first potential binding site in the
CYS3 promoter (position
168
relative to the ATG). As shown in Fig.
3, Xbp1 was able to bind
these sequences.
Moreover, these sequences are bound with a higher
affinity than the
consensus binding site obtained by site selection,
as judged by the
band intensity of the retarded DNA (Fig.
3).
Interestingly, the
homologies of these new binding sites to the
Xbp1 consensus binding
site derived from site selection (
29)
was limited. The only
completely conserved bases were within the
central core of the Xbp1
binding site (
29), yet those tested
(asterisks in Fig.
2)
bind Xbp1 with higher affinity than does
the consensus binding site.
This suggests that the central core
of the Xbp1 binding site is the
most important part of the binding
site, but that bases outside of the
core are also critical to
achieve high-affinity binding. This
conclusion warrants the modification
of the consensus binding site to
[(a/c)CTCGA(g/a)(g/a)(a/g)n (a/g)]
(Fig.
2).
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FIG. 3.
Binding of Xbp1 to CLN3 and CYS3
promoter sequences. Gel retardation experiments used recombinant
His6-Xbp1 and the following oligonucleotides as probes and
competitors: con (consensus binding site determined by binding site
selection), mut (six positions of the consensus binding site are
mutated), CL3 (CLN3 promoter binding site), and CY3
(CYS3 promoter binding site, position 168). Equal amounts
(counts per minute) of probe were used for all binding reactions.
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XBP1 is expressed during meiosis.
Chromosomal
regions that undergo unusually high levels of recombination during
meiosis are termed meiotic recombination hot spots. One of the
best-characterized hot spots in S. cerevisiae is the
CYS3 locus (9, 34). In the promoter of
CYS3 there are two sites where DSBs are observed during
meiosis, CYS3-I (270) and CYS3-II (160) (34). The
CYS3-II site also overlaps exactly the first potential Xbp1 binding
site in the CYS3 promoter. Considering the growing number of
examples where meiotic recombination and transcription or transcription
factor binding are linked (32), we investigated whether
XBP1 is expressed during meiosis and therefore could
potentially contribute to CYS3-II hot spot activity.
First, we searched the
XBP1 promoter for elements known to
be involved in regulating gene expression during meiosis.
Interestingly,
the
XBP1 gene is adjacent to and divergently
transcribed from
the
SGA1 gene, a meiosis-specific
glucoamylase (
22).
SGA1 is
induced in sporulating
cells and repressed by the presence of
nutrients (
22).
Within the 770-bp region between
SGA1 and
XBP1 there is an upstream activating sequence (UAS) and a negative
regulatory element, identified in the region between
620 and
519
relative to
XBP1 (
22) (Fig.
4), which are able to confer
meiosis-specific induction to an heterologous promoter. Overlapping
these elements there is a sequence bearing strong homology to
the MSE
consensus, which is also sufficient to direct sporulation-specific
expression (
36) (Fig.
4). This sequence was recently shown
to
be specifically bound by Ndt80 (
11). In addition, there
are
sequences nearer to the
XBP1 coding sequence which have
homologies
to other meiotically regulated elements: UAS
SPS4
(
19) and the
T
4C element of
IME2
(
5) (Fig.
4).
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FIG. 4.
The SGA1-XBP1 intergenic promoter. Previously
identified promoter elements responsible for stress-induced expression
of XBP1 are shown as gray boxes for stress response elements
(STREs), AP1 recognition element (ARE), and heat shock element (HSE) as
indicated. The region from 620 to 519 (relative to the
XBP1 ATG) contains sequences previously characterized as a
negative regulatory element (NRE) and UAS for the expression of
SGA1 (22). Black boxes represent homologies of
XBP1 promoter sequences to UAS elements known to direct
meiosis-specific expression: MSE, UASSPS4, and
T4C (see text). Positions of promoter elements are drawn to
scale; numbers indicate distances (in nucleotides) relative to the
XBP1 ATG.
|
|
To see if these or other sequences modulate
XBP1 expression
during meiosis, we measured the expression of
XBP1 in SK-1
cells
(
1,
20), which can be induced to go through meiosis
and sporulation
efficiently and synchronously. After shifting these
cells into
sporulation medium, we took aliquots every 2 h for up
to 16 h.
After 24 h, the cells were shifted to rich medium
(YEPD) and additional
samples were taken for 4 h. RNA was purified
from these samples,
and the
XBP1 transcript and other
meiosis-specific transcripts
were monitored by Northern blot
hybridization (Fig.
5B). As expected,
XBP1 was not expressed during mitotic growth in
presporulation
medium (
29). It was detectable after the
culture was shifted
to the starvation conditions of sporulation medium,
but after
8 h, a very strong induction of
XBP1
transcript and protein occurred;
this level was maintained for at least
24 h, by which time spore
formation was complete (Fig.
5B and C).
FACS analyses over this
time course indicated that the strong induction
of
XBP1 occurred
well after the completion of meiotic DNA
synthesis, which was
achieved after 6 h (Fig.
5A) (
30,
37). When the cells were
transferred to rich medium, which
induces germination and subsequent
mitoses,
XBP1 RNA and
protein dropped to barely detectable levels
within 2 h.
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FIG. 5.
Expression of XBP1 during meiosis. An SK-1
derivative strain (NKY 278) was shifted at time zero from
presporulation medium (YEPA) to sporulation medium. Progression through
meiosis was monitored, and aliquots were taken every 2 h for up to
16 h. After 24 h, the culture was shifted to rich medium
(YEPD) and monitored for another 4 h. (A) Progression through
meiosis was monitored by measuring the DNA content at the indicated
time points by FACS analysis and analyzing the data with the CellQuest
program. The positions of peaks representing 2n or 4n DNA content are
labeled. (B) Total RNA was prepared, and the expression of
XBP1, ACT1, DMC1, NDT80,
SGA1, DIT1, and SPS100 was analyzed by
Northern blotting. The blots were hybridized simultaneously or
sequentially. The ethidium bromide (EtBr)-stained gel is shown at the
bottom. Exposure times were 8 h for the XBP1-specific
hybridization and 1 h for the SGA1-specific
hybridization. (C) Protein extracts were prepared from the same time
points and subjected to immunoprecipitation using a polyclonal antibody
directed against Xbp1. Precipitated proteins were analyzed by Western
blotting using the same antibody. The 72-kDa Xbp1 band is shown.
|
|
The relative timing of
XBP1 transcription was also compared
to that for the transcription of other known meiosis-specific
genes.
For each of the four classes of genes, early, middle, mid-late,
and
late, we chose one representative and analyzed its expression
on
Northern blots. As shown in Fig.
5B,
DMC1 is an early gene
which is expressed between 2 and 8 h,
NDT80 shows
mid-sporulation
expression which peaks between 6 and 10 h, and
DIT1 shows a mid-late
expression pattern that peaks between
8 and 12 h.
SPS100 is expressed
even later, between 12 and 24 h. The timely appearance of these
genes is as described
previously (
4,
7,
11,
25). By
this analysis,
XBP1
defines a new pattern of late sporulation-specific
expression, in that
it is induced at the same time as mid-late
genes like
DIT1,
but differs from them in that its transcript
persists throughout the
time course (24
h).
The expression pattern for Xbp1 that we observe is not consistent with
that provided by the microarray analysis that has been
carried out on
sporulating cells (
10). However, our Northern
blot analysis
was repeated three times, and the
XBP1 transcription
pattern
correlates well with our Western blot analysis of Xbp1.
The microarray
data represent a single experiment in which a low
degree of synchrony
was achieved and samples were taken for only
11.5 h. The
progression of meiotic S phase in our experiment cannot
be directly
compared to data from the array experiment, but we
note that some early
and mid-early genes, i.e.,
DMC1 and
NDT80,
show a
prolonged pattern of expression in the array data compared
to ours.
Therefore, it is possible that Xbp1 induction occurred
after their last
time
point.
The same blot was hybridized with a probe for
SGA1, which is
adjacent to
XBP1 and is divergently transcribed (Fig.
4).
Interestingly,
there are clear differences in the timing and strength
of expression.
SGA1 is expressed at a much higher level (see
the legend to Fig.
5B) than
XBP1 and is induced at least
2 h before
XBP1 is turned
on. It is also turned off by
14 to 16 h, when
XBP1 mRNA is still
high (Fig.
5B).
Although
SGA1 has been classified as a late sporulation
gene
(
30), in this strain it is expressed just after
NDT80 is
turned on and before
DIT1 is induced
(Fig.
5B). This timing is
more consistent with
SGA1 being a
mid-late sporulation
gene.
The expression of
XBP1 during meiosis is roughly correlated
with the appearance of DSBs at meiotic recombination hot spots
(
14). In
S. cerevisiae, nearly all meiotic
recombination hot
spots have been mapped to promoter-containing
regions, and for
some of them the binding of transcriptional activators
has been
shown to be necessary (
49). In the case of the
CYS3 hot spot,
which coincides with an Xbp1 binding site, it
is possible that
the binding of Xbp1 is important. To address this
question, we
compared the appearance of DSBs at the
CYS3
locus. As shown in
Fig.
6B, DSBs peak at
8 to 10 h after transfer to sporulation
medium. Since deletion of
XBP1 does not change the timing or the
frequency of DSBs at
this locus, we conclude that binding of Xbp1
to sites in the
CYS3 promoter is not necessary for the formation
of DSBs.
Similar observations have been made for the DSBs at the
ARG4
locus, where Gcn4 binding sites were identified but a disruption
of the
GCN4 locus displayed no changes in the levels of
ARG4 gene
conversion (
41).
CYS3 is
another example where enhanced recombination
is coincident with but not
dependent on a transcription factor
binding site. This inference
supports the view that another feature,
perhaps chromatin
accessibility, is more important for hot spot
formation
(
32).
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FIG. 6.
Formation of meiotic DSBs at the CYS3 locus
in diploid rad50S strains. (A) Map of the CYS3
region indicating positions of the DSBs and the EcoRI probe
(gray bar) used to detect the DSBs. (B) rad50S cells wild
type for XBP1 (BY2533) or with a deletion of XBP1
(xbp1) (BY2534) were sporulated; at the indicated times,
chromosomal DNA was prepared, digested with HindIII, and
analyzed by Southern blot hybridization with an EcoRI
fragment from the CYS3 locus. The asterisk marks the
unbroken HindIII fragment. The two adjacent mapped
CYS3 DSBs ( 110 bp apart) are not resolved on this blot.
|
|
Xbp1 represses genes late in meiosis.
The deletion of
XBP1 in haploid cells has no obvious phenotype
(29). To determine whether XBP1 is required for
meiosis and sporulation, we constructed a diploid Xbp1-deficient SK-1
strain and compared its sporulation to that of an isogenic
XBP1 strain. These strains showed equivalent timing of DNA
replication, as judged by FACS analyses (data not shown), and tetrads
dissected after 24 h of sporulation showed high spore viability
and normal kinetics of germination (data not shown). The timing of
meiosis II in the two strains was also identical as judged by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (DAPI)
staining (Fig. 7). In contrast, when the
percentage of spore-containing asci was scored, we found that ascus
formation was considerably delayed in xbp1 cells and was
almost 25% less efficient (Fig. 7). These results indicate that Xbp1
is important for events which occur after meiosis and before or during
spore formation. DNA synthesis and the meiotic divisions occur with normal kinetics. In wild-type cells, asci form about 2 h later and
are 50% complete after 8 h, which is about when Xbp1 expression is induced. When Xbp1 is deleted, half-maximal ascus formation is
delayed another 2 to 3 h.
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FIG. 7.
Spore formation in XBP1 and
xbp1 strains. The SK-1 derivative strains BY1633
(XBP1) and BY2388 (xbp1) were shifted at time
zero from presporulation medium (YEPA) to sporulation medium. Aliquots
were taken at indicated time points, and cells were fixed in 3.7%
formaldehyde. The percentage of sporulation (percent asci; solid lines)
was determined by scoring the asci with two or more spores in each
ascus under a microscope. Most asci contained four spores under the
conditions used. The percentage of meiosis II nuclei (dashed lines) was
determined by DAPI staining and fluorescence microscopy. The percentage
of cells having completed meiosis II was calculated by dividing the sum
of tri- and tetranucleate cells by the total number of cells. The
standard error is only shown if it is larger than 2%.
|
|
Since Xbp1 is highly induced during the latter half of gametogenesis,
we investigated the effect of loss of Xbp1 on the meiotic
expression
pattern of the Xbp1 target genes that were identified
by Xbp1
overproduction and differential display (reference
29 and Fig.
1). As shown in Fig.
8, the
CLN1 transcript level
is
very low throughout meiosis and
CLN3 is immediately
repressed
after the shift to sporulation medium. By contrast, in
Xbp1-deficient
cells,
CLN1 mRNA is much higher, particularly
after 12 h.
CLN3 also shows a moderate derepression at
late time points. As previously
shown (
11), the mRNA for the
B-type cyclin gene
CLB2 increases
transiently after the
completion of meiotic S phase (6 to 9 h)
(Fig.
5) in wild-type
cells. In
xbp1 cells,
CLB2 is induced to
the
same extent but is maintained at a higher level at the later
time
points compared to wild-type cells. Another potential Xbp1
target gene,
CYS3, is induced early in meiosis, and its mRNA level
declines after 6 h. As with
CLB2, this decline is
absent in Xbp1-deficient
cells (Fig.
8). The same behavior was observed
for
SMF2, whose
mRNA declines gradually in wild-type cells
but remains high in
Xbp1-deficient cells. Thus, it appears that Xbp1
promotes the
decline of message levels for all five of its putative
target
genes during meiosis and has its greatest effect on
CLN1.
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FIG. 8.
Transcriptional effects of XBP1 during
meiosis. The SK-1 derivative strains BY1633 (XBP1) and
BY2388 (xbp1) were treated as for Fig. 5. Aliquots were
taken at indicated time points, and total RNA was prepared. The
expression CLN1, CLN3, CLB2,
CYS3, SMF2, DMC1, NDT80,
SGA1, DIT1, XBP1, ACT1, and
TCM1 was analyzed by Northern blotting. The expression of
ACT1 and the ribosomal protein gene TCM1 served
as the loading control. The ethidium bromide (EtBr)-stained gel is
shown at the bottom. The RNAs from wild-type and xbp1
cells were probed on the same blot, and blots were exposed for the same
amount of time; in producing the figure, the image was split to make a
clear distinction between the two strains.
|
|
Since spore formation is delayed in
xbp1 cells, we
analyzed the expression of the meiosis-specific genes
DMC1,
NDT80,
SGA1,
and
DIT1 in the same time
course (Fig.
8). The expression patterns
of the early and middle genes
(
DMC1,
NDT80, and
SGA1) show no
difference in timing, although
NDT80 is expressed at a
higher
level in Xbp1-deficient cells (Fig.
8). This is of interest
because
there is a consensus for Xbp1 binding in the
NDT80
promoter region.
Xbp1-dependent repression of
NDT80 could
not have been observed
in our differential display screen, as
NDT80 is not expressed
at all in mitotic cells. Thus, it is
possible that Xbp1 exerts
some repressive effect upon
NDT80,
but the fact that the overall
pattern of
NDT80 expression is
not changed indicates that Xbp1
is not the only regulator of this gene.
The late gene,
DIT1, is
expressed for at least twice as long
in
xbp1 cells compared to
wild-type cells. This prolonged
period of
DIT1 expression may
be a result of the elevated
levels of
NDT80 transcript in
xbp1 cells,
since
DIT1 is likely to be an Ndt80-regulated gene (
10,
11). There are no obvious Xbp1 binding sites in the
DIT1 promoter.
CLN1 may be the key target of Xbp1-dependent repression
late in gametogenesis.
The G1 cyclin gene
CLN1 shows the strongest transcriptional derepression when
sporulating wild-type cells are compared to Xbp1-deficient cells. This
prompted us to investigate whether this inability to repress
CLN1 could be responsible for the defects in spore formation
observed in xbp1 cells. CLN1 is not required for meiosis in budding yeast (15), but high-level expression of CLN1 has a dramatic deleterious effect upon spore
formation (12). Thus, we deleted CLN1 in the
xbp1 and XBP1 SK-1 strains and quantitatively
compared the kinetics of ascus formation in these strains. The first
unexpected finding was that loss of CLN1 function caused a
3-h delay and reduced the efficiency of sporulation in SK-1 cells by
about 25% (Fig. 9). This indicates a
previously unknown requirement for CLN1 function for
efficient sporulation. Second, there was no further delay when
CLN1 and XBP1 were both deleted. Since Xbp1
represses CLN1 transcription late in sporulation, one
possible explanation of these results is that CLN1 is
required early in this process, but as the spores are formed,
CLN1 must be repressed. This late repression of
CLN1 expression may be the critical function of Xbp1 during
sporulation, because when CLN1 is deleted, loss of Xbp1
function does not cause a further delay or defect in spore formation.
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FIG. 9.
Spore formation in the cln1 strains. Spore
formation in the cln1 strains BY2677 (cln1)
and BY2678 (xbp1 cln1) was measured as described for
Fig. 7. For comparison, the percentage of sporulation for a wild-type
strain is shown as a dashed line. The standard error is only shown if
it is larger than 2%.
|
|
| |
DISCUSSION |
Nutrient limitation starts a transcriptional cascade which
triggers meiosis and gametogenesis, or sporulation, in a diploid yeast
cell. In budding yeast, 1 of 6 genes (about 1,000) shows significant
changes in transcript level during sporulation. Half of these
transcripts are induced, and half are repressed (10). Discrete waves of transcriptional induction have been characterized as
being early, middle, mid-late, and late in gametogenesis (reviewed in
references 24 and 30), but Xbp1
is the first sporulation-specific transcriptional repressor to be identified.
Xbp1 is highly induced during meiosis and defines a new class of
sporulation-specific genes. XBP1 message is present at a low
level immediately after the shift to starvation conditions; then it
undergoes a burst of synthesis at about the same time as the mid-late
genes and remains high throughout spore maturation, which occurs long
after the mid-late genes are turned off. The low-level expression of
XBP1 at the very beginning of the meiotic program is most
likely a response to the starvation conditions of these cultures, since
XBP1 is induced by limiting for glucose (29). The
mid-late burst of XBP1 transcription could be activated through the adjacent UASSPS4 or the other, more
distal elements that reside within the XBP1 promoter region,
but these elements induce transient expression which typically begins
earlier than that of XBP1 (19, 30). Thus, it is
plausible that a novel promoter element or an interplay between these
known meiotic elements contributes to the pattern of XBP1
transcription. The XBP1 message may also be more stable than
other meiotic transcripts, and this could contribute to its unique
expression pattern.
XBP1 is adjacent to and divergently transcribed from another
sporulation-specific gene, SGA1, and yet the SGA1
and XBP1 transcript levels begin to rise at different times,
attain very different levels, and persist for different intervals
during the sporulation program. These genes are clearly not
coordinately expressed, even though they are separated by only 750 bp
of DNA which includes several potential promoter elements that are
known to be bidirectional. It would be of interest to know how this
differential gene expression is achieved.
Xbp1-dependent repression of cyclins during gametogenesis.
Our
overexpression studies suggested that three cyclin genes
(CLN1, CLN3, and CLB2) and two other
genes (CYS3 and SMF2) are likely targets for
Xbp1-mediated repression. This is indeed the case, as all five of these
transcripts are repressed during the late stages of gametogenesis, and
Xbp1 activity is required for this repression. The advantage, if any,
of repressing CYS3 and SMF2 is unclear, but it is
striking that three cyclins, each of which has a distinct function in
the mitotic cell cycle, are actively repressed during the late stages
of the meiotic cell cycle.
The role of G
1 cyclins in the onset of meiosis in yeast has
only recently been addressed (
12,
15).
CLN3 is
highly expressed
in presporulation medium, when cells are growing very
slowly,
but it is immediately downregulated when the cells are
transferred
to starvation conditions which trigger meiosis. This
immediate
drop is not Xbp1 dependent, but its radical nature suggests
that
CLN3 may be actively repressed upon the shift to
sporulation conditions.
Indeed, Colomina et al. (
12) have
shown that
CLN3 overproduction
prevents meiosis, probably by
interfering with the activation
of Ime1, which is a transcription
factor required to start the
meiotic cell cycle. Moreover,
cln3 cells enter premeiotic S phase
more rapidly than
wild-type cells, and cells in which
CLN3 is
downregulated
and
IME1 is induced undergo meiosis spontaneously
(
12). These results are all consistent with the view that
Cln3
kinase activity antagonizes the meiotic program. However, the
fact
that
cln3 cells behave differently than
CLN3
cells in meiosis
indicates that Cln3 is expressed to a significant
extent under
these circumstances. One possibility is that Cln3 plays a
limited
role early in the process and then is actively repressed at
later
times.
The roles of the other two G
1 cyclin genes,
CLN1
and
CLN2 have not been investigated in detail. Strains
carrying deletions
of either
CLN1 or
CLN2 still
form spores (
15), but the timing
and efficiency of this
process had not been studied previously.
We have found that
cln1 cells display a significant delay and
a 25% reduced
efficiency of spore formation. This is a rather
dramatic phenotype for
a gene product which is considered to be
largely redundant with Cln2
and clearly indicates a positive role
for Cln1 in the sporulation
pathway. This is also consistent with
the fact that the two primary
activators of
CLN1 transcription,
Swi4 and Swi6, are also
present during meiosis and deletion of
either gene results in reduced
spore viability (
26). In agreement
with Leem et al.
(
26), we observe a strong transient induction
of Swi4
transcript levels early in the meiotic program and note
that the
SWI4 promoter contains a consensus URS1 element, which
is
known to induce many meiotic transcripts (B. Mai and L. Breeden,
unpublished data).
CLN1 transcript levels appear constant
throughout
meiosis and spore maturation in wild-type cells. However, in
cells
with a deletion of
XBP1, we detect a strong
derepression of
CLN1 during the late stages of this
process.
CLN1 may be the critical target of Xbp1-dependent
repression in meiosis.
In addition to the finding that Xbp1
actively represses a subset of the budding yeast cyclins during
gametogenesis, we find that diploids lacking Xbp1 are slower and less
efficient in this developmental process. The simplest explanation of
this delay is that the inability to repress one or more of the Xbp1
target genes is deleterious to spore formation. The CLN1
transcript is low throughout the meiotic cycle, but in the absence of
Xbp1 this transcript is highly derepressed through the late stages of
sporulation when Xbp1 is normally expressed. This led us to test
whether the delay of sporulation that occurs in xbp1
strains could be due to this ectopic expression of Cln1. The results
clearly indicate that loss of Xbp1 does not cause a further delay of
sporulation in cln1 cells. This is consistent with the
view that Xbp1's critical function in sporulation is to repress
CLN1. However, we also found that deletion of
CLN1 itself reduces sporulation efficiency, so we must
assume that CLN1 also has a role in this process. The simplest interpretation is that CLN1 has a function in
sporulation, but that the high levels of CLN1 that arise
late in the absence of Xbp1 is deleterious.
To achieve a maximum efficiency during the process of gametogenesis,
Cln1 has to be regulated very tightly at two points during
meiosis and
spore formation. It has to be expressed at a low level
early during
meiosis and has to be shut off or at least maintained
at that low level
later, at a time when spores are formed. If
one of these points of
regulation is lost, by deleting either
CLN1 or
XBP1, proper timing of spore formation is abrogated. Thus,
we conclude that Xbp1's primary role is to keep
CLN1
repressed
late in
meiosis.
In aggregate, our results suggest that the G
1 cyclin Cln1
and its transcriptional repressor Xbp1 play important but nonessential
roles in sporulation. We hypothesize that Cln1/Cdc28 kinase activity
early in the sporulation program increases the efficiency of this
pathway, but the same activity is deleterious if it persists through
the late stages of gametogenesis. Since spores are more resistant
to
unfavorable environmental conditions, the integrity of this
developmental process and the speed with which cells can recover
from
this state and return to the mitotic cycle represent considerable
evolutionary advantages. Xbp1 and Cln1 are two of many cellular
activities that contribute to this
coordination.
| |
ACKNOWLEDGMENTS |
We thank Nancy Kleckner, Harvard University, for providing the
SK-1 strains and Ron Reeder, Fred Hutchinson Cancer Research Center,
for the pFA-kanMX6 plasmid. Special thanks are offered to Ingrid Wolf,
Christoph Sachsenmaier, and other members of the laboratory for
numerous constructive suggestions and discussions.
This work was supported by NIH grant GM41073 to L.B. and by a Deutsche
Forschungsgemeinschaft postdoctoral fellowship to B.M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fred Hutchinson
Cancer Research Center, Division of Basic Sciences, A2-168, 1100 Fairview Ave. N. Seattle, WA 98109-1024. Phone: (206) 667-4484. Fax:
(206) 667-6526. E-mail: lbreeden{at}fred.fhcrc.org.
| |
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Abstract
Introduction
