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Molecular and Cellular Biology, August 2000, p. 5766-5776, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Activation of the Kss1 Invasive-Filamentous Growth
Pathway Induces Ty1 Transcription and Retrotransposition in
Saccharomyces cerevisiae
Antonin
Morillon,
Mathias
Springer, and
Pascale
Lesage*
UPR 9073 du CNRS, Institut de Biologie
Physico-Chimique, F-75005 Paris, France
Received 17 December 1999/Returned for modification 15 February
2000/Accepted 28 April 2000
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ABSTRACT |
Using a set of genomic TY1A-lacZ fusions, we show that
Ste12 and Tec1, two transcription factors of the Kss1 mitogen-activated protein kinase (MAPK) cascade activate Ty1 transcription in
Saccharomyces cerevisiae. This result strongly suggests
that the invasive-filamentous pathway regulates Ty1 transcription.
Since this pathway is active in diploid cells, we suspected that Ty1
transposition might occur in this cell type, despite the fact that this
event has been never reported before (unless activated by heterologous
promoters such as that of GAL1). We demonstrate here that
constitutive activation of the invasive-filamentous pathway by the
STE11-4 allele or by growth in low-nitrogen medium induces
Ty1 transcription and retrotransposition in diploid cells. We show that
Ty1 retrotransposition can be activated by STE11-4 in
haploid cells as well. Our findings provide the first evidence that Ty1
retrotransposition can be activated by environmental signals that
affect differentiation. Activation of the Kss1 MAPK cascade by stress
is known to cause filament formation that permits the search for
nutrients away from the colonization site. We propose that activation
of Ty1 retrotransposition by this cascade could play a role in adaptive
mutagenesis in response to stress.
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INTRODUCTION |
Retrotransposons are a class of
mobile genetic elements that move via an RNA intermediate. They are
structurally and functionally related to retroviruses. Five different
families, Ty1 to Ty5, have been identified in Saccharomyces
cerevisiae (6). The Ty1 copia-like
retrotransposon is the most abundant (31). Thirty-two copies
of this element are present in the sequenced genome of the strain S288C
(33).
Ty1 contains an internal coding region of 5.3 kb, flanked by two long
terminal repeats (LTR) of 0.33 kb (6). The internal domain
contains two overlapping open reading frames, TY1A and TY1B, analogous to the retroviral gag and
pol genes, respectively. Ty1 is transcribed from LTR to LTR
by RNA polymerase II, the resulting transcript serving as a template
for both translation and reverse transcription. Ty1 preferentially
integrates next to RNA polymerase III promoters (18), but
less-frequent insertions into or upstream of genes transcribed by RNA
polymerase II have been reported.
Ty1 insertions have been shown to activate the expression of genes such
as ADH2, CYC7, CAR1, CAR2,
DUR1, and DUR2 (6). In this case, Ty1
transcription is always divergent from the activated gene, and the
transcription start site of the target gene is generally not modified
upon Ty1 insertion. These mutant alleles, called ROAM (for regulated
overproducing alleles responding to mating) behave as haploid-specific
genes, since their mRNA levels decrease in a/
diploid
cells (24). Their expression is reduced in ste7,
ste11, ste12, and tec1 mutants
(19, 25, 34). The Ste11 and Ste7 proteins are a
mitogen-activated protein kinase (MAPK) kinase kinase (MEKK), and a
MAPK kinase (MEK), respectively, and Ste12 and Tec1 are transcription
factors acting downstream of Ste7 and Ste11. These four proteins are
involved in an invasive-filamentous developmental pathway that has been
described in both haploid and diploid cells (41) (Fig.
1). In diploid cells, nitrogen starvation
induces a switch from growth as a single-cell form to chains of
elongated cells (called filaments or pseudohyphae) which invade the
agar surface (30). In haploid cells, activation of this
pathway leads to agar invasion, but the inducing environmental signals
are not known (49). This mode of growth is believed to
enable cells to forage for nutrients at a distance from their initial
colonization site, under adverse conditions. The invasive-filamentous pathway shares several signaling components with the mating pathway (38). However, their specificity of action is ensured by two distinct MAPKs that limit cross talk between these pathways. The Kss1
MAPK acts in the invasive-filamentous pathway, and the Fus3 MAPK is
dedicated to the mating pathway (15, 42). Tec1 ensures another level of specificity (40, 41). In the
invasive-filamentous pathway, Tec1 binds cooperatively with Ste12 to a
DNA site called FRE (filamentation- and invasion-responsive element).
In the mating pathway, Ste12 binds to DNA, as a homomultimer or as a
heteromultimer with Mcm1.
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FIG. 1.
MAPK pathways regulating invasive-filamentous and mating
developments. The invasive-filamentous pathway is depicted on the left,
and the mating pathway is shown on the right. Ste5 binds Ste11, Ste7,
and Fus3 proteins and is required for pheromone-induced signaling
(41). FRE, filamentation- and invasion-responsive element;
PRE, pheromone-responsive element; Pbox, Mcm1 binding site. In the
invasive-filamentous pathway, Ste12 binds to FRE cooperatively with
Tec1. Ste12 acts in the mating pathway by binding to DNA as a
homomultimer or as a heteromultimer with Mcm1.
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Two regions of Ty1 are responsible for haploid-specific and
STE-dependent regulation of ROAM mutant expression (12). The first region, encompassing nucleotides 384 to 433, is responsive to
Ste7 and Ste12 activation (11). It contains an FRE site to which Tec1 and Ste12 have been shown to bind in vitro (2,
40). The second region (nucleotides 815 to 927) contains both
Mcm1 and a1/2 repressor complex binding sites that confer
enhancer and diploid control activities, respectively (26).
Some findings suggest that Ty1 expression is regulated as in ROAM
mutants: mRNA levels decrease 10-fold in diploid cells (21)
and are reduced in ste7, ste11, or
tec1 mutants (19, 34). The lower levels of Ty1
mRNA in these mutants, along with the presence of an FRE site in the
Ty1 sequence, suggest that Ty1 transcription might be regulated by the
invasive-filamentous pathway. The use of Ty1 FRE as a tool to identify
components of the invasive-filamentous pathway strengthens this
hypothesis (40, 42). However, for this identification, Ty1
FRE was used as an upstream activating sequence of reporter genes,
while in the Ty1 retrotransposon, FRE is located downstream of the TATA
box, in the TY1A coding sequence. This unusual location
might prevent activation of Ty1 transcription by the
invasive-filamentous cascade. Consistent with an abnormal regulation,
the Ste12 activator was proposed to act as a repressor of Ty1
expression (10). Thus, we decided to analyze further the
regulation of Ty1 expression and retrotransposition by the
invasive-filamentous pathway.
In this study we show that the transcription of most native Ty1
elements is activated by Ste12 and Tec1 and is regulated by the
invasive-filamentous pathway. Our results also indicate that Ty1
transcription and retrotransposition can be activated in diploid cells
by this pathway. Since invasive-filamentous growth is a response to
environmental stress, we propose that activation of Ty1
retrotransposition by this pathway may help survival of stress conditions by creating adaptive mutations.
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MATERIALS AND METHODS |
Yeast strains and media.
Yeast strains used in this work are
described in Table 1. All derivatives of
S288C contains the flo8-1 recessive allele that impairs
filamentous growth (39). Since the FLO8 gene is a
target of the cyclic AMP signaling pathway that regulates pseudohyphal growth independently from the Kss1 MAPK cascade (47), the
flo8-1 mutation will not affect activation of the Kss1 MAPK
in S288C. Although some derivatives of S288C strains were reported to
be kss1
(22), we found that the
FYBL1-23D and FY839 strains, both isogenic to the S288C strain used as
the source of DNA for the European Union Yeast Genome Sequencing
Program (55), were wild types for KSS1. The
evidence came from the ability of the STE11-4 allele to
activate FUS1-lacZ fusion in FYBL1-23D, FYBL1-23D
fus3
and FYBL1-23D kss1 strains but not in
FYBL1-23D fus3 kss1 (A. Morillon and P. Lesage, unpublished data). Identical results were obtained by Elion et
al. with strain W303 after activation with pheromones instead of
STE11-4 (22). These results were interpreted as
meaning that the strain is wild type for both FUS3 and
KSS1.
The
1278b derivatives L5684 and LV148 (obtained by disrupting the
HIS3 open reading frame with
TRP1 in L5685) were
used to
make diploid cells when filamentous growth needed to be checked
(
39). LV101 and LV105 were obtained by disrupting the
STE12 open reading frame in FYBL1-23D and FY1679-28C,
respectively.
LV126 and LV128 were obtained by disrupting
TEC1 in FYBL1-23D
and LV50, respectively. Null alleles of
STE12 and
TEC1 were obtained
by one-step gene
replacement using PCR fragments of the
TRP1 gene
amplified
with long primers containing 5' and 3' sequences of
STE12
and
TEC1, respectively (
1). Gene replacements
were checked
by PCR analysis. Similarly, LV237 and LV247 were obtained
by one-step
gene replacement of
KSS1 in FYBL1-23D and FY839,
respectively,
using a PCR fragment of the
KanMX gene with 5'
and 3' sequences
of
KSS1 (
43). All gene
replacements were checked by PCR
analysis.
Strain LV150, which carries a
ROAM-his34-1
allele was constructed in two steps. First, we constructed strain LV69
by replacing
the
LYS2 locus in FYBL1-23D with a
lys2::his34 allele as described
by
Z. Qian (
48). In a second step, LV150 was selected as an
His
+ prototroph upon galactose induction of pGTy1-H3
(kindly provided
by J. Boeke) in LV69, as described previously
(
5). PCR analysis
indicated that a Ty1 element was
integrated approximately 150
bp upstream of
his34 in LV150 and that Ty1 and
his34 were oriented
divergently.
Strains JC297 and JC242, used in the transposition assays, were kindly
provided by M. J. Curcio (
13,
16).
Yeast transformations were performed by the LiAc method (
1).
Yeast cells were grown in rich (YPD), synthetic complete (HC),
synthetic minimum (SD), and synthetic low-ammonium (SLAD) media
(
1,
30). Unless otherwise stated, 2% glucose was used as
the carbon
source.
Construction of 31 strains each carrying a TY1A-lacZ
fusion at a different Ty1 locus.
Construction of these strains
will be described in detail elsewhere (unpublished data). Briefly,
strain FYBL1-23D was transformed with a DNA fragment carrying
lacZ fused in frame to TY1A (at coordinate 1571 of Ty1-H3 [4]) and located upstream of the
URA3 gene followed by sequences of TY1B. In the
'ty1a'-'lacZ-URA3-'ty1b' construct, the 'ty1a' sequence comes from Ty1-H3 (coordinates 1144 to
1571). Since the first 1 kb of Ty1 is sufficient to produce high levels of Ty1 transcripts (6), the use of a ty1a
fragment starting at coordinate 1144 should not modify the
transcriptional level of the Ty1 element fused to lacZ in
the recombinants. To identify which Ty1 element carried the fusion,
Ura+ recombinants obtained upon transformation were
analyzed by PCR using 32 oligonucleotides, each specific to the region
upstream of one Ty1 locus and an oligonucleotide specific to
lacZ. The synthesis of a DNA fragment by PCR amplification,
using one oligonucleotide specific to a Ty1 element and the
lacZ oligonucleotide, allowed us to identify which Ty1
element carried the TY1A-lacZ fusion in the recombinant. In
parallel, the DNA of the recombinants was digested with several
restriction enzymes that do not cut in TY1A but do cut
downstream of lacZ to perform Southern blot analysis. The
hybridization of a single fragment to a probe specific to lacZ indicated a single integration event. The size of the
labeled fragment, compared to the size predicted with the restriction map of the 32 Ty1 loci, allowed us to confirm which Ty1 element carried
the TY1A-lacZ fusion in these strains.
Null alleles of
STE12 and
TEC1 were obtained in
these strains by one-step gene replacement using PCR fragments of the
TRP1 gene as described above with LV101 and LV126.
Similarly, a null
allele of
KSS1 was obtained in these
strains by one-step gene
replacement using PCR fragments of
KanMX, as described for
LV247.
Construction of GAL1p-TY1A(PR1)-lacZ fusion.
The
GAL1p-TY1A(PR1)-lacZ fusion is a TY1A-lacZ fusion
at Ty1-PR1 in which the U3 region of 5' LTR has been replaced by the GAL1 promoter. This construct was done in several steps.
First, a BamHI-EcoRI fragment containing the
TRP1 sequence (coordinates 
80 to 728) and obtained by PCR
amplification was ligated with the 756-bp
EcoRI-XhoI fragment of pGTy1-H3 (5)
containing the GAL1 promoter and with pRS426 digested with
BamHI and XhoI (9). In the resulting
pAM3 plasmid, TRP1 and GAL1p are divergent. In parallel, a fragment encompassing the 606 to 58 region upstream of
Ty1(PR1) in the genome of S288C was PCR amplified, and a
BamHI site was created adjacent to position 58 (position
+1 corresponds to the first nucleotide of 5' LTR). This PCR product was
ligated to the 2.9-kb BamHI-NcoI fragment of pAM3
in order to place the fragment homologous to the region upstream of
Ty1(PR1) adjacent to TRP1-GAL1p. The final construct,
upstream Ty1(PR1)-TRP1-GAL1p-R, was then obtained by
high-fidelity PCR amplification using a primer specific to the region
upstream of Ty1(PR1) (coordinates 403 to 383) and a primer specific
to GAL1p with a 5' extension homologous to the R region of
Ty1 LTR. This construct was then integrated in the FYBL1-23D derivative
containing the TY1A(PR1)-lacZ fusion by homologous
recombination. Finally, the replacement at Ty1(PR1) was checked by PCR.
The GAL1p-TY1A(DR3)-lacZ fusion was obtained by a similar strategy.
Plasmids.
The pAM7 plasmid (DPS1-lacZ URA3 CEN)
was constructed in two steps. First, pAM6 was constructed by cloning in
Yep353 (45), a 2-kb BamHI-HindIII
fragment carrying the first 400 bp of DPS1, as well as 1.6 kb of upstream regulatory sequence (kindly provided by G. Eriani).
Then, the 5.9-kb BamHI-NcoI fragment of pAM6
carrying the DPS1-lacZ fusion was ligated with the
corresponding 3.5-kb fragment of pRS316 (52), yielding pAM7.
Plasmids pSL974 (FUS1-lacZ URA3, 2µm; kindly provided by
G. Sprague), p11-4/HIS3 (STE11-4 HIS3 CEN), pSL1509
(STE11-4 URA3 CEN), and pFRE(Ty1)-lacZ [FRE(Ty1)-lacZ URA3, 2µm; generously provided by G. Fink] have already been
described (32, 40, 42).
Northern blot analysis.
Total RNA was extracted from 10-ml
cultures grown at 22°C to mid-log phase as described earlier
(3) without the 70% ethanol RNA washing step. For each
strain, 2 µg of RNA was loaded onto a 1% agarose-0.5× TBE gel,
unless otherwise stated. The size-fractionated RNA was blotted onto a
Hybond-N membrane (Amersham), and hybridization was performed as
recommended by the supplier. Probes against Ty1, Ty1his3AI
(i.e., HIS3), lacZ, and ACT1 were
generated by random priming. The Ty1 probe was derived from a region of
Ty1 nonhomologous to Ty2 (coordinates 3137 to 3682 in Ty1-H3). Results
were quantified on a Molecular Dynamics PhosphorImager with ImageQuant software.
-Galactosidase assays.
For filter lift assays, patches of
strains grown at 30°C on HC-URA plates were replica plated on a fresh
HC-URA plate covered with a 1MM Whatman sterile paper. Replica were
incubated overnight at 30°C. Cells were lysed on filters by three
successive cycles of freezing (10 min at 80°C) and defreezing (10 min at 37°C). Finally, filters were soaked at room temperature in Z
buffer plus X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside; 100 µg/ml) to detect -galactosidase activity. After 2 h, the
reaction was stopped in 1 M NA2CO3.
For liquid assays, precultures were grown at 30°C overnight in HC
medium lacking the appropriate nutrients to maintain selection
for
plasmids. Precultures were diluted 100-fold in selective medium
and
grown at 22°C to mid-log phase. Whole-cell extracts were prepared
as
described earlier (
50) except that Z buffer without
-mercaptoethanol
was used as the extraction buffer. Protein
concentration was determined
by the Bio-Rad assay, and
-galactosidase activity was measured
as described elsewhere
(
44).
-Galactosidase units are expressed
in nanomoles of
2-nitrophenyl-
-
D-galactopyranoside hydrolyzed
per minute
per nanogram of protein. Values are the averages of
at least three
independent measurements. Standard deviations are
<10%.
Transposition assay.
To determine the transposition
frequency, cells were grown at 30°C overnight in HC medium lacking
the appropriate nutrients to maintain selection for plasmids.
Precultures were diluted 50-fold into selective medium and grown for
three generations at 22°C. For each culture, aliquots were plated on
YPD and HC medium lacking histidine to determine the number of
His+ prototrophs.
Assays under nitrogen starvation conditions were done as follows.
Patches were replica plated from SD medium supplemented
with histidine
(high nitrogen) to SD medium supplemented with
histidine (high
nitrogen) and SLAD medium supplemented with histidine
(low nitrogen).
After 7 days of growth at 22°C, patches were replica
plated again on
SD medium plus histidine (for patches on the SD-plus-His
plates) and on
SLAD medium plus histidine (for patches on the
SLAD-plus-His plates) to
ensure nitrogen starvation in the latter
case. After 7 days of growth
at 22°C, replica plates were done
on SD medium (lacking histidine) to
score retrotransposition events
of Ty1
his3AI-270 that result
in His
+ prototrophs. Plates were incubated for 4 days at
30°C.
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RESULTS |
Native Ty1 elements are poorly transcribed in diploid cells.
There are 32 native Ty1 elements in the genome of S. cerevisiae S288C. To study the relative expression level of each
endogenous Ty1 element, we constructed haploid strains each expressing
lacZ under the transcriptional controls of a different Ty1
element at its original locus. Since sequences located in
TY1A are required for Ty1 transcription, lacZ was
fused in frame to the 3' end of the TY1A gene of each native
Ty1 element (see Materials and Methods for details).
Ty1A-lacZ fusions were named according to the Ty1 sequence
annotation by MIPS (http://www.mips.biochem.mpg.de/), i.e.,
TY1A(PR1)-lacZ corresponds to lacZ fused to the
first Ty1 element of chromosome XVI. Thirty-one different strains were
constructed; one native Ty1 copy (Ty1-H) was not successfully fused to
lacZ. The expression of the 31 TY1A-lacZ fusions
was determined in haploid cells using filter lift assays. Most patches
of haploid cells were blue or light blue in the presence of X-Gal, and
five remained white (Table 2). Northern
blot analysis was performed to determine whether the -galactosidase
levels reflected the steady-state level of TY1A-lacZ mRNA in
these strains. Total RNA extracted from three strains yielding blue,
light blue, and white patches, respectively, in the presence of X-Gal
was hybridized to a lacZ probe (Fig.
2). The results indicated a clear
correlation between steady-state levels of TY1A-lacZ
transcripts and the -galactosidase activity estimated in these
strains.
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FIG. 2.
Northern blot analysis of TY1A-lacZ
transcripts in haploid cells. For each strain, 10 µg of RNA was
loaded on gel. Steady-state levels of TY1A(PR1)-lacZ,
TY1A(OL)-lacZ, and TY1A(DR3)-lacZ mRNA were
normalized with ACT1 mRNA. The ratios are given relative to
TY1A(DR3)-lacZ. Details on -galactosidase activity are
given in Table 2. Strains were grown in HC medium lacking uracil.
Strains carrying the TY1A-lacZ fusions are derivatives of
FYBL1-23D.
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To determine whether the
-galactosidase activity reflects the
promoter activity of each fusion, we replaced the promoter
of
TY1A(PR1)-lacZ and
TY1A(DR3)-lacZ with the
inducible
GAL1 promoter,
such that transcription of these
fusions becomes regulated by
galactose availability (see Materials and
Methods). The
-galactosidase
activities of
TY1A(PR1)-lacZ
and
TY1A(DR3)-lacZ fused to the
GAL1 promoter
were similar (150 and 151 U, respectively), while
TY1A(PR1)-lacZ -galactosidase activity was 50-fold higher
than
TY1A(DR3)-lacZ -galactosidase activity when these
fusions were expressed from
their own promoters (26 and 0.5 U,
respectively). Thus, the monitoring
of
-galactosidase activity in
these strains allowed us to assess
the promoter activity of each native
Ty1
element.
In diploid cells, the decrease of total Ty1 mRNA (
21)
suggests that Ty1 expression is repressed, but it is not known if
all
Ty1 elements are regulated identically. To address this issue,
the 31 haploid strains carrying the different
TY1A-lacZ fusions
in
FYBL1-23D were crossed with FY839 to obtain diploid cells.
By comparing
the expression of the 31
TY1A-lacZ fusions in haploid
and
diploid cells with filter lift assays, we observed a general
decrease
of
-galactosidase activity in diploid cells (Table
2).
Most patches
of diploid cells were white. We performed a quantitative
-galactosidase assay for the 10
TY1A-lacZ fusions still
expressed
in diploid cells on plates. Except for the
TY1A(LR4)-lacZ fusion,
which was only slightly repressed in
diploid cells, the
-galactosidase
activity was close to background
for all fusions, indicating that
expression of the corresponding Ty1
elements was repressed in
diploid cells (Fig.
3). As expected, a similar decrease was
observed
for the haploid specific
FUS1-lacZ fusion. Finally,
we compared
the sum of the
-galactosidase activities of the 10 fusions expressed
to a measurable level in diploid cells to the sum of
specific
activities obtained with the 31
TY1A-lacZ fusions
expressed in
haploid cells. A 10-fold decrease of total
-galactosidase activity
was observed in diploid cells (Fig.
3). The
decrease is similar
to that of Ty1 mRNA previously measured in diploid
cells (
21).
These results indicate that expression of all
native Ty1 copies,
with the noticeable exception of Ty1(LR4), is
strongly repressed
in diploid cells.
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FIG. 3.
Expression of native Ty1 elements in haploid and diploid
cells. The figure shows the -galactosidase activity of the
TY1A-lacZ fusions that gave a blue or light-blue color in
filter lift assays of diploid cells. The total is the sum of the
specific activities obtained with the 31 TY1A-lacZ fusions
expressed in haploid cells and in diploid cells. GAL1p-PR1 is a
TY1A-lacZ fusion, in which the U3 region of 5' LTR has been
replaced by the GAL1 promoter at Ty1(PR1) (see Materials and
Methods). +glu., in the presence of glucose; +gal., in the presence of
galactose. FUS1-lacZ is carried on pSL974. Diploid cells
were obtained by mating the FYBL1-23D derivatives carrying different
TY1A-lacZ fusions with FY839.
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To determine whether the dramatic decrease in Ty1 expression was
promoter dependent, we compared the
-galactosidase activity
of
TY1A(PR1)-lacZ, fused to the
GAL1 promoter, in
haploid and
diploid cells grown in the presence of galactose. The
-galactosidase
activity was 300-fold higher in the presence of
galactose than
in the presence of glucose (Fig.
3). However, no
difference in
-galactosidase activity was observed between the
haploid and
diploid cells. A similar result was obtained when the
promoter
of
TY1A(DR3)-lacZ was replaced by the
GAL1 promoter (data not
shown). These results indicate that
repression of the promoter
activity of Ty1 is responsible for the
decrease of Ty1 expression
in diploid
cells.
Ste12 and Tec1 activate Ty1 transcription in haploid cells.
An
increase of Ty1 mRNA in ste12 haploid cells has been
previously reported, suggesting that Ste12 might act as a repressor of
Ty1 transcription (10). This observation is surprising,
since Ste12, which binds in association with Tec1 to Ty1 FRE in vitro, is known to activate FRE-driven expression (40). Therefore, we decided to analyze the role of STE12 on Ty1 transcription
further, by monitoring the -galactosidase activity of several
TY1A-lacZ fusions in wild-type and ste12 strains.
STE12 was disrupted in 10 derivatives of FYBL1-23D carrying
the best-expressed
TY1A-lacZ fusions. The sum of the
expression
of these fusions accounts for 75% of the total in wild-type
haploid
cells (A. Morillon and P. Lesage, unpublished data). In the
absence
of
STE12,
-galactosidase activity diminished
significantly for
all fusions (5- to 17-fold compared to the wild
type), except
for the
TY1A(LR4)-lacZ fusion, where only a
2-fold decrease was
observed (Fig.
4A).
Interestingly, the
TY1A(LR4)-lacZ fusion was
also not fully
repressed in diploid cells (Fig.
3). In control
experiments, expression
of
FUS1-lacZ, which is known to depend
on
STE12
(
32), was dramatically reduced in the presence of the
deletion, whereas expression of
DPS1-lacZ remained constant
(
DPS1 encodes the aspartyl-tRNA synthetase and is
constitutively expressed).
By comparing the sum of the
-galactosidase activities obtained
with the 10
TY1A-lacZ
fusions in the
ste12 strain (i.e., 38.6
U) with the total
activity of the 31 fusions expressed in wild-type
haploid cells (325 U
[Fig.
3]), we calculate an eightfold decrease
in
TY1A-lacZ
expression in the absence of
STE12. A Northern blot
analysis, performed in parallel to compare the total level of
Ty1 mRNA
in wild-type and
ste12 haploid cells, indicated a similar
decrease of total Ty1 transcripts levels in the absence of
STE12 (ninefold, Fig.
4B). The reduction of
-galactosidase activity
for the 10 fusions analyzed, as well as the
lower level of Ty1
mRNA in a
ste12 strain, demonstrate
that
STE12 enhances Ty1 transcription
in haploid cells.
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FIG. 4.
Ste12 activates Ty1 transcription. (A) -Galactosidase
activity determined in haploid STE12 and ste12
cells grown in HC medium lacking uracil. Details on lacZ
fusions are given in the legends to Fig. 2 and 3. DPS1-lacZ
is carried on the centromeric plasmid pAM7. Plasmids were introduced in
FYBL1-23D (STE12) and LV101 (ste12). (B)
Northern blot analysis of steady-state level of Ty1 mRNA normalized
with ACT1 mRNA in FY1679-28C (STE12) and LV105
(ste12) strains grown in YPD. The ratios of total Ty1
mRNA to ACT1 mRNA relative to the STE12 strain
were as follows: STE12, 1.0; ste12, 0.11.
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We also deleted
TEC1 in three strains carrying the
TY1A(JR2)-lacZ,
TY1A(ML2)-lacZ, and
TY1A(PR1)-lacZ fusions, respectively.
As observed in the
absence of
STE12, the
-galactosidase activity
of these
fusions decreased 3- to 10-fold in the absence of
TEC1 (data
not shown). This result is in agreement with already-published
data
indicating a reduction of Ty1 mRNA levels in
tec1 mutant
(
34). Taken together, these results indicate that both
STE12 and
TEC1 activate Ty1 transcription in
haploid
cells.
Activation of the Kss1 MAPK cascade by the STE11-4
allele induces Ty1 transcription in diploid cells.
The positive
role of both Ste12 and Tec1 on Ty1 transcription shown above and the
previous evidence that Ty1 transcription requires Ste7 and Ste11
strongly suggest that Ty1 expression is under the control of the Kss1
invasive-filamentous pathway (references 19 and
25 and Fig. 1). Since this pathway can be activated in diploid cells, we predicted that Ty1 transcription could be similarly stimulated in this cell type. Previous experiments have shown
that FRE-dependent gene expression and filamentation can be induced in
diploid cells by the hypermorphic STE11-4 allele that leads
to constitutive phosphorylation of Kss1 (42). Therefore, we
decided to test whether activation of Ste12 and Tec1 by the invasive-filamentous pathway in the presence of STE11-4
would activate Ty1 transcription. Since Fus3 is not expressed in
diploid cells, STE11-4 does not activate the mating pathway
in these experiments (23, 29).
Ten derivatives of FYBL1-23D carrying different
TY1A-lacZ
fusions, chosen because they are well expressed in haploid cells
but
repressed in diploid cells [except for the
TY1A(LR4)-lacZ fusion], were crossed with FY839. The resulting diploid strains
were
transformed with a plasmid expressing
STE11-4 and a plasmid
vector, respectively. Little
-galactosidase activity was detected
in
the absence of
STE11-4, except for the
TY1A(LR4)-lacZ fusion,
whose expression is not fully
repressed in diploid cells. However,
in the presence of
STE11-4, a significant increase in
-galactosidase
activity was observed for most fusions (Fig.
5A). The sum of the
individual
-galactosidase values for the 10 tested diploid strains
indicates
that the
STE11-4 plasmid causes a 3.8-fold increase
in Ty1
expression (compare 88.5 U obtained in the presence of
STE11-4 with 23 U obtained in the absence of
STE11-4).
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FIG. 5.
Induction of the Kss1 MAPK cascade in the presence of
STE11-4 activates Ty1 transcription. (A) -Galactosidase
activity determined in diploid cells obtained by mating the FYBL1-23D
derivatives carrying different TY1A-lacZ fusions with FY839.
Diploid cells were transformed with the pRS313 vector (control)
(52) or with p11-4/HIS3 (STE11-4) and grown in HC medium
lacking uracil and histidine. Details on lacZ fusions are
given in the legends to Fig. 3 and 4. In FRE(Ty1)-lacZ, the
27-bp Ty1 FRE is located upstream of an enhancerless
CYC1-lacZ reporter gene (40). pFRE(Ty1)-lacZ and
pAM7 (DPS1-lacZ) were introduced in diploid cells obtained
by crossing FYBL1-23D with FY839. (B) -Galactosidase activity
determined in wild-type, kss1/kss1, and
tec1/tec1 diploid cells transformed with
p11-4/HIS3 (plus STE11-4) and grown in HC medium lacking uracil and
histidine. Wild-type diploid cells were obtained by crossing the
FYBL1-23D derivatives carrying the different TY1A-lacZ
fusions with LV50. pFRE(Ty1)-lacZ was introduced in diploid cells
obtained by mating FYBL1-23D with LV50. The
kss1/kss1 diploid cells were obtained by
disrupting KSS1 in the FYBL1-23D derivatives carrying the
different TY1A-lacZ fusions and by crossing the resulting
strains with LV247. Similarly, the
tec1/tec1 diploid cells were obtained by
disrupting TEC1 in the FYBL1-23D derivatives carrying the
different TY1A-lacZ fusions and by crossing the resulting
strains with LV128. pFRE(Ty1)-lacZ was introduced in diploid cells
obtained by crossing LV126 with LV128 (for
tec1/tec1 diploid cells) and LV237 with
VL247 (for kss1/kss1 diploid cells).
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|
To verify that the activation of Ty1 transcription occurred via the
Kss1 invasive-filamentous pathway, we deleted
KSS1 in
three
diploid strains carrying different
TY1A-lacZ fusions and
measured their
-galactosidase activities in the presence of a
plasmid expressing
STE11-4. The three fusions were not
activated
by
STE11-4 in the absence of
KSS1 (Fig.
5B). Similarly, the three
fusions were not activated by
STE11-4 when
TEC1 was deleted (Fig.
5B). Taken
together, our results indicate that
STE11-4 acts on
Tec1 and
Ste12, via the Kss1 MAPK cascade, to activate Ty1 transcription.
The
-galactosidase activity observed with
Ty1(FRE)-lacZ in
the
absence of Kss1 (Fig.
5B) is due to the fact that Kss1 has two
antagonistic activities (Fig.
1) (
42); thus, deletion of
KSS1 leads to an intrinsic activity of Tec1 and Ste12. The
-galactosidase
activity measured in diploid cells transformed with
STE11-4 was
lower than in haploid cells (see Fig.
4A),
suggesting that activation
of Ty1 transcription by Ste12 and Tec1 does
not completely overcome
the repression of haploid specific genes by the
a1/
2 complex
in diploid
cells.
The expression of two fusions,
TY1A(BL)-lacZ and
TY1A(LR4)-lacZ, did not increase in the presence of
STE11-4 (Fig.
5A). Whereas
the Ste12 binding-site of most
native Ty1 elements (
CGTTTCA) differs
from the
consensus site by one nucleotide (
TGTTTCA
[
40]), that
of Ty1(BL) contains two differences
(
CATTTCA) (
Saccharomyces Genome
Database,
http://genome-www.stanford.edu/). Ty1(BL) belongs with
Ty1(MR1) to a subtype of Ty1 elements (
33). Ty1(MR1)
presents
the same modified Ste12 binding site. As for
TY1A(BL)-lacZ,
TY1A(MR1)-lacZ expression was not
activated by
STE11-4 in diploid cells (data
not shown). In
haploid cells,
TY1A(BL)-lacZ expression is high
and
requires Ste12 (Fig.
4A), indicating that Ste12 is still able
to bind
to Ty1(BL) DNA despite the two differences in the consensus
binding
site. However, the lack of activation of
TY1A(BL)-lacZ and
TY1A(MR1)-lacZ fusions by
STE11-4 in diploid
cells suggests
a less-efficient binding of Ste12 to its DNA target
which, in
combination with the lower amount of Ste12 present in diploid
cells, could severely reduce Ste12-mediated activation. With respect
to
TY1A(LR4)-lacZ, the lack of activation by
STE11-4
was expected
since, despite the presence of a normal Ste12
binding-site, its
expression is only partially dependent on Ste12 (Fig.
4A). We
suggest that the specific location of Ty1(LR4), which overlaps
the
HAP1 open reading frame (
Saccharomyces Genome
Database), may
impair its regulation by Ste12: RNA polymerase II
molecules transcribing
HAP1 may facilitate transcription of
Ty1(LR4) without a need for
additional transcriptional
activators.
Activation of the Kss1 MAPK cascade by STE11-4 induces
Ty1 retrotransposition.
We wondered whether activation of Ty1
transcription by the Kss1 MAPK cascade in diploid cells would be
followed by retrotransposition events. Transposition of a genomic Ty1
element under the control of its native promoter can be detected when
the element is tagged with the retrotransposition indicator
his3AI. The transposition frequency of this element is
proportional to the frequency of His+ prototroph formation
(13, 16). We measured the frequency of His+
colony formation in diploid cells obtained by crossing FY839 with
JC297 and JC242, respectively. These diploid strains containing the endogenous Ty1his3AI-270 or Ty1his3AI-242
elements were transformed by the pRS316 vector or by the pSL1509
plasmid carrying STE11-4. While no His+ colonies
were detected with pRS316 transformants, His+ prototrophs
arose in the presence of STE11-4. The frequency of Ty1his3AI-270 or Ty1his3AI-242 retrotransposition
was similar to that found in haploid cells in the absence of
STE11-4 (Table 3). These
results indicate that activation of the invasive-filamentous Kss1 MAPK
cascade induces Ty1 retrotransposition in diploid cells.
We also performed a Northern analysis to compare the amount of
Ty1
his3AI-270 and Ty1 transcripts in the absence and in the
presence of
STE11-4 (Fig.
6).
A fivefold increase in Ty1
his3AI-270 mRNA was detected in
the presence of
STE11-4 in diploid cells,
strongly
suggesting that activation of Ty1
his3AI-270
retrotransposition
is a direct effect of transcriptional activation of
this element
in the presence of
STE11-4 (Fig.
6A, top
panel). Total Ty1 mRNA
levels were also twofold higher in the presence
of
STE11-4 (Fig.
6A, center panel). The increase in
Ty1
his3AI-270 and Ty1 mRNA
levels in the presence of
STE11-4 confirms that induction of the
invasive-filamentous
pathway by
STE11-4 activates Ty1 transcription
in diploid
cells, as shown above with the activation of
TY1A-lacZ expression in the presence of
STE11-4.
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FIG. 6.
Northern blot analysis of total RNA from diploid and
haploid cells in the presence or in the absence of STE11-4.
(A) Strains were transformed with either the pRS316 vector (STE11-4)
or with pSL1509 (+STE11-4) and grown in HC medium lacking uracil.
Haploid and diploid strains are described in Table 3. Probes against
HIS3 and Ty1 were used to detect Ty1his3AI-270
and Ty1 mRNA, respectively. Ty1his3AI-270 and Ty1 mRNA
levels were normalized with ACT1 mRNA. The increase of
Ty1his3AI-270 mRNA levels in the presence of
STE11-4 was 5-fold in diploid cells and 1.6-fold in haploid
cells; that of Ty1 mRNA levels was 2-fold in diploid cells and 1.4-fold
in haploid cells. (B) Segment of a shorter exposure of the same
Northern blot to show the slight increase of Ty1his3AI mRNA
in haploid cells transformed with pSL1509 (+STE11-4).
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|
Interestingly, we found that
STE11-4 significantly enhances
the retrotransposition frequency of the endogenous
Ty1
his3AI-270 and Ty1
his3AI-242 elements in
haploid cells as well (15.5- and
6.4-fold, respectively [Table
3]).
An increase in Ty1
his3AI-270 and Ty1 transcript levels was
detected by Northern analysis in
haploid cells in the presence of
STE11-4 (Fig.
6). Although minor
(1.6- and 1.4-fold,
respectively), this increase suggests that
STE11-4 also
activates Ty1 transcription in haploid cells. Taken
together, these
results show that
STE11-4 activates Ty1 transcription
and
retrotransposition in diploid and in haploid
cells.
Ty1 transcription and transposition are activated in diploid cells
starved for nitrogen.
As nitrogen starvation induces filamentation
of diploid cells (30), we asked whether Ty1 transcription
and transposition would be activated in diploid cells grown in
low-nitrogen medium. Since the flo8-1 recessive mutation
impairs filamentous growth in S288C derivatives, we constructed diploid
cells able to filament by crossing the 1278b derivative L5684, which
is FLO8, with three derivatives of FYBL1-23D carrying
different TY1A-lacZ fusions. Production of filaments on
low-nitrogen medium confirmed that nitrogen starvation induces the
invasive-filamentous pathway in these diploid strains (data not shown).
To determine if Ty1 transcription was activated upon nitrogen
starvation, we measured the -galactosidase activity in the three
diploid strains grown either in high- or low-nitrogen medium. In
nitrogen-starved cultures, expression of the three TY1A-lacZ
fusions [as well as the FRE(Ty1)-lacZ control] increased
severalfold (Fig. 7A), whereas expression
of DPS1-lacZ did not. We conclude that Ty1 transcription is
activated in diploid cells upon nitrogen starvation.
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FIG. 7.
Nitrogen starvation activates Ty1 transcription and
transposition. (A) -Galactosidase activity determined in diploid
cells obtained by crossing the FYBL1-23D derivatives carrying different
TY1A-lacZ fusions with L5684. Cells were either grown in SD
medium (High N, high nitrogen) or in SLAD medium (Low N, low nitrogen).
Details on lacZ fusions are given in the legends to Fig. 4
and 5. (B) Retrotransposition of the endogenous
Ty1his3AI-270 element. Patches correspond to four different
diploid strains obtained by crossing JC297 with LV148. SD plates are
shown after 4 days of incubation at 30°C. On the left are shown
diploid cells replica plated onto the SD plate after growth on SLAD
plus histidine (nitrogen-starved diploid cells), and on the right are
shown diploid cells replica plated onto an SD plate after growth on SD
plus histidine (nitrogen-fed diploid cells). The nonrandom localization
of His+ colonies probably results from higher growth at the
edge of the patches.
|
|
We also analyzed the transposition frequency of the endogenous
Ty1
his3AI-270 element in nitrogen-starved diploid cells. We
constructed a diploid strain containing the endogenous
Ty1
his3AI-270 element and a complete deletion of
HIS3 on both chromosomes by
mating JC297 with the
1278b
derivative LV148. This strain was
unable to grow in the absence of
histidine. Patches of diploid
cells containing the
Ty1
his3AI-270 element were grown at 22°C
either on high
(SD)- or low (SLAD)-nitrogen plates supplemented
with histidine.
Filaments were detected after several days of
growth on low-nitrogen
plates, indicating that the invasive-filamentous
pathway was activated
upon nitrogen starvation. Patches were subsequently
replica plated on
SD medium lacking histidine to identify retrotransposition
events.
His
+ prototrophs appeared only with the patches of cells
grown on
low-nitrogen plates, indicating that Ty1
his3AI-270
retrotransposition
is activated in nitrogen-starved diploid cells (Fig.
7B). In conclusion,
the activation of
TY1A-lacZ
transcription in nitrogen-starved
diploid cells along with the
transposition of Ty1
his3AI-270 shows
that stimulation of the
invasive-filamentous pathway by nitrogen
starvation induces Ty1
transcription and retrotransposition in
diploid
cells.
Ty1 insertions can confer regulation by the invasive-filamentous
pathway to adjacent genes.
ROAM mutants can easily be selected
using the promoterless his34 allele, which is
activated in haploid cells by upstream Ty1 insertions (7).
Because of the presence of the FRE sequence in Ty1 and based on the
experiments described above, we predict that expression of
ROAM-his34 alleles should be under the control of the invasive-filamentous pathway. As a consequence, their
expression, which is constitutive in haploid cells and repressed in
diploid cells, should be induced in diploid cells when the
invasive-filamentous pathway is activated.
We failed to detect retrotransposition events upstream of
his34 in diploid cells upon activation of Ty1
retrotransposition
by the invasive-filamentous pathway in the presence
of
STE11-4,
presumably because
his34 is not a preferential target for Ty1
integration. Thus, we decided to first construct a haploid strain
carrying a
ROAM-his34 mutation by
transpositional induction using
pGTy1-H3 (see Materials and Methods).
Using this approach, His
+ prototrophs of LV69 that arise at
a frequency of 5 × 10
7 per cell (data not shown)
were obtained. One such strain, LV150,
was crossed with FY839 to obtain
a diploid strain carrying
ROAM-his34-1.
This
strain was then transformed with a plasmid expressing
STE11-4 or with a plasmid vector as a control. Transformants
containing
the plasmid vector were unable to grow in the absence of
histidine,
whereas His
+ diploid cells grew in the presence
of
STE11-4 (Fig.
8). In
conclusion,
the activation of
ROAM-his34-1
expression in the presence of
STE11-4 indicates that, in
diploid cells, induction of the invasive-filamentous
pathway can
activate the expression of genes located in the proximity
of a Ty1
element, as is the case in ROAM mutants.
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FIG. 8.
Activation of ROAM-his34-1
allele in diploid cells in the presence of STE11-4. Diploid
cells containing the ROAM-his34-1 allele were
transformed either with the pRS316 vector (control) or with pSL1509
(+STE11-4) and grown to saturation in SD medium supplemented with
histidine liquid cultures. Transformants were assayed for His
expression by spotting serial dilutions on SD medium and for growth by
spotting on SD-plus-histidine plates. Haploid and diploid cells
containing the promoterless his34 allele were
unable to grow in the absence of histidine (data not shown). Diploid
cells were obtained by crossing LV150 with FY839.
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|
| |
DISCUSSION |
To study the transcriptional regulation of the Ty1 elements in
their native location, we constructed 31 strains, each carrying a
lacZ chromosomal fusion expressed from the transcriptional
signals of a different Ty1 element (Morillon and Lesage, unpublished
data). We focused our analysis on the 10 best-expressed
TY1A-lacZ fusions since their global expression amounts to
75% of the total, thus giving a representative picture of the
regulation of Ty1 expression. Our experiments support the view that the
Kss1 MAPK invasive-filamentous pathway activates Ty1 transcription and
that stress-like conditions, i.e., constitutive activation of this
cascade or nitrogen starvation, can induce Ty1 transcription and
retrotransposition in diploid cells.
The Kss1 invasive-filamentous pathway activates Ty1
transcription.
First, we found that TY1A-lacZ
transcription in haploid cells requires Ste12 and Tec1, two
transcriptional activators acting downstream of the
invasive-filamentous pathway and that bind cooperatively to the FRE
site (2, 40). In agreement with the activation of
TY1A-lacZ transcription by Ste12, our Northern blot analysis indicated that Ty1 mRNA levels decrease severalfold in the absence of
STE12 (Fig. 4). In ROAM mutant alleles, in Ty1 FRE reporter gene constructs, as well as in the TEC1 and FLO11
genes (6, 46, 51), FRE is located upstream of the TATA box.
In the case of Ty1, it is located 157 bp downstream of the
transcription start site, in the TY1A open reading frame.
Our results indicate that Ty1 FRE behaves as a downstream activating
sequence of endogenous Ty1 elements, as suggested by previous
experiments which showed that a region encompassing Ty1 FRE is required
to activate transcription of a Ty1 element carried on a plasmid
(28). Thus, Ste12 and Tec1 can activate transcription, when
recruited to FRE sites located upstream or downstream of a TATA box.
In addition to the FRE site, other downstream activating sequences have
been identified in Ty1 (
57). Downstream repressing
and
activating sequences are also present in Ty2 (
27,
37).
Since
sequences regulating Ty transcription should be located
within the
retrotransposon to be kept upon retrotransposition,
the presence of
regulatory sequences downstream of the TATA box
of Ty1 and Ty2
retrotransposons suggests that this particular
location could reflect
an adaptation to size restraints of the
LTR on these elements. The
existence of such restraints is supported
by the observation that
increasing the length of the LTRs inhibits
Ty1 retrotransposition
(
35). Since the 5' LTR needs to be an
active promoter unlike
the 3' LTR, the presence of regulatory
sequences downstream from the 5'
LTR may also be the best way
to specify promoter activity to this
LTR.
A second piece of evidence that the invasive-filamentous signaling
pathway regulates Ty1 transcription is that the constitutive
STE11-4 allele, shown to activate both filamentation and
FRE-dependent
gene expression (
42) induces
TY1A-lacZ transcription in diploid
cells (Fig.
5A). This
result is supported by the two- and fivefold
increases in Ty1 and
Ty1
his3AI-270 mRNA levels, respectively,
observed in diploid
cells in the presence of
STE11-4 (Fig.
6).
The Ste11 MEKK
acts upstream of Fus3 and Kss1 MAPK to regulate
both the mating and
invasive-filamentous pathways (
41). Activation
of Ty1
transcription in diploid cells depends exclusively on the
invasive-filamentous pathway since activation of
TY1A-lacZ
expression
by
STE11-4 requires Tec1 and Kss1 (Fig.
5B),
which are specific
to this pathway, and also because
FUS3 is
not expressed in diploid
cells (
23,
29). In agreement with
the activation of Ty1 transcription
by the filamentous pathway but not
by the mating pathway,
TY1A-lacZ transcription was activated
in diploid cells upon nitrogen starvation,
which has been shown to
induce filamentous growth (Fig.
7A).
The Kss1 invasive-filamentous pathway activates Ty1
transposition.
In diploid cells, constitutive activation of the
invasive-filamentous pathway by STE11-4 causes not only Ty1
transcription but also transposition of the endogenous
Ty1his3AI-270 and Ty1his3AI-242 elements (Table
3). Activation of this pathway by nitrogen starvation also induces
Ty1his3AI-270 retrotransposition (Fig. 7B). The activation of Ty1 retrotransposition is probably the direct consequence of Ty1
transcriptional activation by the invasive-filamentous pathway, since
previous results suggest that increased Ty1 mRNA levels cause an
increase in transposition frequency (16, 17). However, it
remains possible that STE11-4 also activates Ty1
retrotransposition at both transcriptional and posttranscriptional
steps in diploid cells.
The presence of
STE11-4 enhances Ty1
his3AI-270
retrotransposition frequency in haploid cells as well (Table
3). This
enhancement
may a priori be due to the activation of either the mating
or
the invasive-filamentous pathway or both. Two results seem to
exclude the involvement of the mating pathway. First, activation
of the
mating pathway by pheromones inhibits Ty1 retrotransposition
(
56). Second, Fus3, the MAPK of the mating pathway, was also
shown to repress Ty1 retrotransposition (
13). Interestingly,
both inhibitions occur at the posttranscriptional step. Therefore,
we
speculate that
STE11-4 activates Ty1 retrotransposition via
the Kss1 invasive-filamentous pathway in haploid cells, as in
diploid
cells. Our results show that
STE11-4 stimulates
Ty1
his3AI-270 retrotransposition 15-fold (Table
3) in
haploid cells. Under
these conditions, Ty1
his3AI-270 mRNA
levels increase 1.6-fold
(Fig.
6), and transcription of some
TY1A-lacZ fusions are 2-fold
higher (Morillon and Lesage,
unpublished data). The lack of proportionality
between the increase in
Ty1
his3AI-270 transcription and transposition
might indicate
that
STE11-4 acts at a posttranscriptional level
to activate
Ty1 retrotransposition in haploid cells, unless a
limited increase in
transcription might be sufficient to observe
larger effects on
transposition. Posttranscriptional controls
of Ty1 retrotransposition
have already been demonstrated for the
Fus3 MAPK and the nucleotide
excision repair-transcription factor
TFIIH subunits SSL2 and RAD3
(
13,
36).
We also noticed that the transposition frequency of
Ty1
his3AI-270 in diploid cells activated by
STE11-4 and in unactivated
haploid cells was similar, while
Ty1
his3AI-270 mRNA levels were
higher in haploid cells
(Table
3 and Fig.
6). This lack of proportionality
between Ty1 mRNA
levels and transposition frequency may be due
to the existence of
additional negative posttranscriptional control(s)
in haploid cells
that are not effective in diploid cells. Since
FUS3 is not
expressed in diploid cells, the inhibition of Ty1
retrotransposition by
Fus3 is specific to haploid cells and may
be responsible for this
difference (
13,
23,
29). Alternatively,
this difference may
simply reflect the fact that the potential
targets for
retrotransposition are twice as abundant in diploid
cells as in haploid
cells.
Conte et al. have proposed that Fus3 negatively regulates the Ty1 life
cycle in haploid cells by destabilizing Ty1 virus-like
particles
(
13). More recent results from the same authors showed
that,
in addition to posttranscriptional effects, deletion of
FUS3
leads to a 2.5-fold increase in Ty1 mRNA (
14). We also
found
that the expression of some
TY1A-lacZ fusion was twofold
stimulated in
fus3 strains (Morillon and Lesage,
unpublished
data). Given that
fus3 mutants are hyperinvasive
and result in
an increase of FRE driven expression (
42), we
propose that
FUS3 also inhibits Ty1 retrotransposition by
limiting the activation
of Ty1 transcription by the Kss1 MAPK
invasive-filamentous
pathway.
Activation of Ty1 retrotransposition by the invasive-filamentous
pathway may be an adaptive response to stress.
Our results provide
the first evidence that Ty1 retrotransposition can be activated by
environmental signals that affect cellular differentiation, such as
nitrogen starvation. We propose that activation of Ty1
retrotransposition by these signals is an adaptive response to stress.
Upon nutrient limitation, diploid cells form filaments, which enable
them to search for nutrients away from their colonization site. The
Kss1 MAPK signaling pathway involved in the induction of this
differentiation process also activates Ty1 transcription and increases
Ty1 transposition to new sites. In some cases, Ty1 integration will
lead to cis activation of cellular genes by Tec1 and Ste12,
which might confer a selective advantage to the cell. This adaptive
response to stress could be highly relevant, since diploid cells are
predominant in nature. To strengthen this hypothesis, we found that Ty1
elements can provide the signals required for activation by the
invasive-filamentous pathway to adjacent genes in diploid cells (Fig.
8).
Activation of Ty1 transcription and retrotransposition has already been
observed in haploid cells exposed to DNA-damaging
agents, which
represent another kind of stress (
8). Similarly,
UV
irradiation of
Escherichia coli cells stimulates
intermolecular
transposition of IS
10 in an
SOS-stress-response-dependent process
(
20). In plants,
retrotransposons that are largely quiescent
during development are also
activated by stress-like wounding
or pathogen attacks (
53).
Identification of retrotransposon-derived
sequences flanking plant and
mammalian genes suggest that some
host gene expression may depend on
transposon signals (
54).
Activation of retrotransposition by
stress might thus be a general
way for genomes to
evolve.
| |
ACKNOWLEDGMENTS |
We are very grateful to C. Condon, S. Gangloff, J. Smith, and P. Stragier for critical reading of the manuscript. We thank members of
our laboratory for stimulating discussions and especially C. Sacerdot
for providing strain LV150. We thank J. Curcio, B. Dujon, G. Faye, and
F. Winston for kindly providing strains. Our thanks also go to J. Boeke, G. Eriani, G. Fink, and G. Sprague for sending us plasmids.
This work was supported by a grant from the CNRS (UPR 9073). A.M. was a
recipient of a Docteur Ingénieur fellowship from the CNRS.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: UPR 9073 du
CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie
Curie, F-75005 Paris. Phone: 33 (1) 58-41-51-25. Fax: 33 (1)
58-41-50-20. E-mail: lesage{at}ibpc.fr.
| |
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Molecular and Cellular Biology, August 2000, p. 5766-5776, Vol. 20, No. 15
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Abstract
Introduction
