(Received for publication, February 18, 1997, and in revised form, April 21, 1997)
From the Department of Biochemistry and Biophysics
and Center for Gene Research and Biotechnology, Oregon State
University, Corvallis, Oregon 97331 and the § Laboratory of
Yeast Genetics, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
Mlu1 cell cycle box (MCB) elements are found near
the start site of yeast genes expressed at G1/S.
Basal promoters dependent on the elements for upstream activating
sequence activity are inactive in swi6 yeast. Yeast were
screened for mutations that activated MCB reporter genes in the absence
of Swi6. The mutations identified a single complementation group.
Functional cloning revealed the mutations were alleles of the
TRR1 gene encoding thioredoxin reductase. Although deletion
of TRR1 activated MCB reporter genes, high copy expression
did not suppress reporter gene activity. The trr1 mutations
strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle
box)-containing reporter genes, but also weakly (3-fold) stimulated
reporter genes that lacked these elements. The trr1
mutations did not affect the level or periodicity of three endogenous
MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and
TRX2 recapitulated the stimulatory effect of
trr1 mutations on MCB reporter gene activity. Conditions
expected to oxidize thioredoxin (exposure to
H2O2) induced MCB gene expression, whereas
conditions expected to conserve thioredoxin (exposure to hydroxyurea)
inhibited MCB gene expression. The results suggest that thioredoxin
oxidation contributes to MCB element activation and suggest a link
between thioredoxin-oxidizing processes such as ribonucleotide
reduction and cell cycle-specific gene transcription.
Mlu1 cell cycle boxes or MCBs1 (consensus ACGCGTNA) are found in the upstream region of budding yeast genes encoding replication enzymes and other proteins preferentially synthesized at the G1/S boundary of the cell cycle (1). Structurally similar elements called Swi4/Swi6 cell cycle boxes or SCBs (consensus CACGAAAA) are found in the upstream region of the HO endonuclease gene, which is also expressed at G1/S (2). Deletion and site-directed mutagenesis has shown that MCB/SCB elements in the upstream regions of TMP1, CDC9, POL1, CLN2, SWI4, and HO are required for efficient gene expression (2-7). Attachment of MCBs or SCBs to basal promoters fused upstream from reporter genes shows that these elements possess G1/S-specific UAS activity (2-4).
Band shift assays using wild type and mutant yeast extracts show that MCBs and SCBs bind a complex containing the transcription factors Swi6 and either Mbp1 or Swi4 (8-11). Band shift assays using purified proteins or in vitro translation products suggest that Swi4 and Mbp1 provide the primary DNA recognition function and that Swi6 enhances the affinity of the complex for its target (10, 12, 13). More recently, another complex that binds SCBs but does not contain Swi6 or Swi4 has been reported (14).
Although the periodicity of MCB-containing genes in vivo and
the majority of MCB binding activity in vitro is dependent
on an intact MBP1 gene (10), the idea that Mbp1 regulates
MCBs whereas Swi4 regulates SCBs may be inaccurate. Overexpression of
SWI4 in swi6 yeast activates MCB reporter
genes in vivo (15) and binding of Swi4 to SCBs in
vitro is efficiently competed by MCB oligonucleotides (12, 13).
Thus, Swi4 may have a role in recognizing and regulating both types of
cell cycle box elements.
The mechanism linking START and MCB gene induction at G1/S
is unresolved. In considering how Swi6 could be activated by START, it
has been noted that Swi6 is a phosphoprotein in vivo,
contains several potential Cdc28 phosphorylation sites, and can be
phosphorylated by human Cdc2 immune complexes in vitro (8).
However, site-directed mutations that eliminate all the potential Cdc28
phosphorylation sites in Swi6 do not eliminate MCB gene periodicity
(16). SWI4 is maximally expressed at G1/S and
contains functional MCB sites in its upstream region (7), suggesting
that Swi4 autostimulation may be a key process in inducing MCB gene
transcription at G1/S. However, constitutive expression of
SWI4 from a heterologous promoter does not eliminate
G1/S gene periodicity (17). MCB binding activity measured
in band shift assays is mildly periodic, with peak levels at
G1/S (4). However, the presence of MCB binding activity, at
least as measured in vitro, is not sufficient for MCB gene expression, as MCB binding activity is abundant in -factor-arrested cells, even though such cells show repressed MCB gene expression (4).
In summary, although important cis- and
trans-acting elements have been identified, the actual
biochemical link connecting START and G1/S-specific
transcription remains elusive.
To search for additional gene products that participate in activating
MCB genes following START, we screened for mutations that allowed
efficient expression of MCB reporter genes in swi6 yeast.
Analysis of the mutations revealed that thioredoxin reductase represses
MCB reporter gene expression, and suggests a model whereby thioredoxin
oxidation may contribute to increased transcription of certain genes at
G1/S.
The 2µ-based plasmids containing the
MCB/LacZ and mutMCB/LacZ reporter genes were
described previously (4), where they were referred to as pLG178.3M
and pLG
178.3mut, respectively. MCB/LacZ contains three
MluI sites, separated and flanked by XhoI sites,
and cloned into the XhoI site at CYC1 base
178
(with respect to the start codon) in plasmid pLG
178 (18). In
mutMCB/LacZ, the MCB consensus ACGCGT was mutated to ACtaGT,
which destroys UAS activity in vivo and band shifting
activity in vitro (4). The plasmid pBd177, which consists of
a 3-kb HindIII/Bgl2 SWI6 fragment
cloned into the LEU2-marked 2µ-based vector pZUC, was obtained from L. Breeden (Fred Hutchinson Cancer Research Center). The
LEU2-marked, 2µ-based libraries YL1, YL2, and YL3
libraries (19) were obtained from P. Bartel (Cold Spring Harbor
Laboratories). The LEU2-marked,
ARS/CEN-based library YpH1 was obtained from P. Hieter (Johns Hopkins University).
The plasmid pMCB/HIS3 was constructed in two steps. First, a
317-base pair fragment containing three tandemly linked MCB elements and the CYC1 basal promoter was generated by PCR using
XbaI-linearized MCB/LacZ plasmid as template and
the oligonucleotides 5-CTAAACTCACAAATTAGAG and
5
-CGGGATCCTGTGTATTTGTGTTTGG as primers. The latter primer introduced a
terminal BamHI site 3
to CYC1 base
14 (with
respect to the start codon). The PCR product was cut with
BamHI and inserted into pRS305 vector (20) that had been cut
with XbaI, filled-in with T4 DNA polymerase and cut with
BamHI, thus creating an interim plasmid containing the
MCB/CYC1 promoter. A second PCR product, extending from
7
to +813 with respect to the HIS3 start codon was generated
using EcoRI-linearized pRS303 (20) as template and the
oligonucleotides 5
-CGGGATCCGGCAAAGATGACAGAGC and
5
-GCCGTCGACGCGCGCCTCGTTCAGAATG as primers. The first primer introduced
a terminal BamHI fragment 5
to HIS3 base
7,
and the second primer introduced a SalI site 3
to
HIS3 base +813. The second PCR product was cut with
BamHI and SalI and inserted into a vector
prepared by cutting the interim plasmid with BamHI and
SalI. Functionality of the MCB/CYC1 promoter and
HIS3 coding region in the resulting plasmid,
pMCB/HIS3, was confirmed by transforming
HpaI-linearized pMCB/HIS3 into W303-1a using
LEU2 selection and confirming that transformants grew in the
absence of histidine and the presence of 50 mM
3-amino-1,2,4-triazole (ATZ).
Strains are listed in Table I.
Except for differences specified in the text or tables, strains were
isogenic to W303. To obtain a swi6:TRP1 strain
that lacked an integrated ho-LacZ gene and
unidentified ade and met mutations, BY600 was
mated to W303-1
. Diploids were sporulated, and dissected tetrads
were assayed for
-galactosidase and nutritional auxotrophies.
Tetrads yielding two Trp+
-galactosidase-negative
colonies and two Trp
-galactosidase-positive colonies
were used to identify
swi6:TRP1 yeast that had
lost the ho-LacZ gene. From these, strains MY1 and MY2 were selected based on the additional criteria of red color
(indicating the ade2 mutation was unaccompanied by other ade mutations) and methionine prototrophy. Strain MY10 was
derived by transforming BY600 with HpaI-linearized
pMCB/HIS3 and selecting for leucine prototrophy. Integration
at leu2 was confirmed genetically by linkage analysis.
|
Strain MY196, which carried a trr1:HIS3 disruption
mutation, was derived using the method of Baudin et al.
(21). Strain MY183, which was homozygous for the his3
200
deletion mutation, was transformed with a PCR fragment containing the
intact HIS3 gene sandwiched between the first 38 and last 34 nucleotides of the TRR1 protein coding region. The PCR
primers were
5-ATGGTTCACAACAAAGTTACTATCATTGGTTCAGGTCCTGATGCGGTATTTTC and
5-TTCTAGGGAAGTTAAGTATTTCTCAGCATCCAAAGCTGTCTGTAAGCGGATGC. DNA from
His+ transformants was analyzed by a Southern blot assay to
determine whether TRR1 was disrupted. MY183 was derived by
crossing YM2061 (MATa ade2-101 ura3-52 LEU2:GAL1/LacZ
his3
200 metT lys2-801) (obtained from M. Johnston, Washington
U.) to MY179 (MAT
ade2 ura3 leu2 trp1 his3
200 metT
ho:LacZ), which itself was derived by crossing YM2061 to MY171
(MAT
leu2 ura3 trp1 metS ho:LacZ). MY171 was derived by
crossing CG378 (MATa ade5 ura3-52 leu2-3 trp1-289) (4)
with MY43.
Strains with lys2 mutations were selected by growth on appropriately supplemented ammonium sulfate-free YNB agar containing 0.2% aminoadipate, and confirmed by showing that lysine prototrophy was restored by transforming with the LYS2-containing plasmid p8LYS2 (S. Sedgewick, National Institute for Medical Research, London).
Standard yeast genetic techniques were used for tetrad dissections and random spore preparations (22). For lys2 heterozygotes, spores were plated on aminoadipate plates, to select against residual diploid cells. For cells carrying trr1:HIS3 alleles, it was necessary to plate spores on YEPD.
Mutagenesis and Isolation of trr1 MutantsEthylmethylsulfonate (EMS) mutagenesis was done as
described by Rose et al. (22). In a pilot experiment,
treatment with 30 µg/ml EMS gave optimal results, reducing viability
by 50% and increasing the frequency of aminoadipate-competent clones
25-fold above the control level of 105/viable cell. To
isolate mutants, 108 MY10Z cells were either treated with
30 µg/ml EMS or mock-treated, and 2 × 106 cells
were spread on each of five 10-cm plates containing YNB agar
supplemented with adenine, methionine, and 10 mM ATZ. An activated MCB/HIS3 reporter gene produces sufficient
imidazole dehydratase activity to result in ATZ-resistance. By 2.5 days after plating, EMS-treated and mock-treated cells gave 50 and 8 ATZ-resistant colonies/plate, respectively. When corrected for the
number of viable cells plated in each group (determined by spreading
aliquots on histidine-supplemented plates), the frequency of
ATZ-resistant cells was 125 × 10
5 in the
EMS-treated population and 6 × 10
5 in the
mock-treated population. All 250 ATZ-resistant clones from the EMS
treatment group were patched to selective plates and assayed for
MCB/LacZ reporter gene activation by filter
-galactosidase assay. Sixty-four gave blue color in the assay, and
of these, 30 were randomly selected for further analysis.
RNA was isolated from
yeast using glass beads and hot phenol (24). RNA concentration was
determined by A260, assuming 1 OD = 40 µg/ml. RNA (2-10 µg) was denatured and fractionated by electrophoresis through 1% agarose, 2.2 M formaldehyde
gels as described by Lehrach et al. (25) except that 10 mM MOPS, 4 mM sodium acetate, 0.5 mM EDTA was used as buffer. Gels were rinsed 5 min with
water, stained 5 min with 1 µg/ml EtBr, rinsed 15 min with water, and
blotted overnight to buffer-equilibrated GeneScreen (DuPont) using
10 × SSC (1 × SSC = 60 mM NaCl, 15 mM sodium citrate, pH 7) as transfer buffer. After
UV-cross-linking (1200 J using a Stratalinker), blots were rinsed in
2 × SSC, prehybridized 3 h at 42 °C in 5-10 ml of
Stark's buffer (50% formamide, 5 × SSC, 25 mM
sodium phosphate, pH 6.5, 0.02% each of bovine serum albumin, Ficoll
400, and polyvinylpyrrolidone, and 250 µg/ml salmon sperm DNA), and
hybridized in 5-10 ml of 4:1 Stark's buffer:dextran sulfate
containing 3 × 104 Bq of radiolabeled probe. Probes
were labeled with (32P)dCTP (3000 Ci/mM, NEN
Life Science Products) using a Random Priming DNA Labeling System kit
(Life Technologies, Inc.) as described by the manufacturer, except that
incorporated radioactivity was isolated by chromatography through a
5-ml Sephadex G50 column equilibrated with 1 × TES buffer (10 mM Tris, pH 7.6, 10 mM EDTA, 1% SDS).
Hybridization probes used were: RNR1, 2.6-kb
EcoRI fragment from pSE738 (S. Elledge, Baylor College of
Medicine); SWI4, 3.2-kb HindIII fragment from
p3-24 (cloned from YL3 library)2;
SWI6, 1.9-kb HindIII/EcoRI fragment
from pBd177 (L. Breeden); TMP1, 1.3-kb
HindIII/EcoRI fragment from pEM54 (3);
LEU2, 2.1-kb EcoRI fragment from pEM54;
TRR1, 1.9-kb EcoRI/XhoI fragment from p29 (cloned from YL1 library by complementation of the
trr1-21 mutation). Blots were washed twice for 5 min at
room temperature with 2 × SSC, 0.1% SDS, and three times for 15 min at 50 °C with 0.1 × SSC, 0.1% SDS. Washed blots were
exposed 1-4 days to x-ray film or PhosphorImager plate. Plates were
analyzed using a model PSI486 PhosphorImager and ImageQuant software
(Molecular Dynamics). Prints were prepared using Adobe Photoshop,
version 2.5.1 (Adobe Systems) and ClarisWorks, version 3 (Claris)
software.
Southern blots were done essentially as described for Northern blots
except that agarose gels containing 1 × TAE (100 mM
Tris, pH 7.5, 100 mM acetate/acetic acid, 10 mM
EDTA) were used and BA85 nitrocellulose (Schleicher & Schuell) was used
instead of GeneScreen. To confirm trr1:HIS3
disruption, transformant DNA digested with
HindIII/EcoRI was blotted and probed with a
radiolabeled 1.9-kb EcoRI/XhoI TRR1
fragment. To confirm nondisruptional tagging of the TRR1
locus, DNA digested with EcoRI was blotted and probed with a
radiolabeled 2.1-kb Bgl2/XhoI TRR1
fragment.
For liquid -galactosidase
assays, exponentially growing yeast were harvested by centrifugation,
resuspended in 250 µl of breakage buffer (100 mM Tris, pH
8, 1 mM dithiothreitol, 20% glycerol), and transferred to
1.5-ml microcentrifuge tubes on ice. After adding 500-µm glass beads
(Sigma) to the meniscus and 12 µl of 40 mM
phenylmethylsulfonyl fluoride, yeast were disrupted by intermittent vortexing and chilling. Lysate was siphoned to fresh tubes and clarified by microcentrifugation for 15 min, and aliquots of the supernatant were diluted with breakage buffer to 90 µl and mixed with
800 µl of Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, adjusted to pH 7), 2 µl of 12 M
-mercaptoethanol, and 180 µl 13 µM
o-nitrophenyl-
-D-galactoside. After 30 min at 30 °C, reactions were stopped by addition of 450 µl of 1 M Na2CO3 and absorbance at 420 nm
was determined to measure the extent of ONP production, assuming 1 OD = 222 nmol/ml. The protein concentration of clarified lysates
were determined by the Bradford method (26), using bovine serum albumin
as standard.
For filter -galactosidase assays, yeast colonies were replica-plated
to Whatman no. 1 filters, frozen in liquid N2, thawed, laid
in a Petri plate on a filter presoaked with 2 ml of Z buffer, 2 µl of
12 M
-mercaptoethanol, and 50 µl of 40 mg/ml
5-bromo-4-chloro-3-indolyl-
-galactopyranoside in Me2SO,
and incubated at 30 °C until blue color developed (1-2 h).
To identify mutations that activated MCB reporter genes in
swi6 yeast, strain MY10Z, which carried an integrated
MCB-dependent HIS3 reporter gene
(MCB/HIS3) and an episomal MCB-dependent
LacZ reporter gene (MCB/LacZ) was mutagenized
with EMS and plated on medium lacking histidine and containing ATZ.
Cells that formed colonies on ATZ were assayed for
-galactosidase
using a filter assay. Sixty-four out of 250 ATZ-resistant colonies gave
blue color. Thirty ATZ-resistant,
-galactosidase-positive mutants were randomly selected for further characterization. The mutants were
grown in liquid culture and lysates were assayed quantitatively for
-galactosidase activity. Table II shows
-galactosidase levels in the mutants, arranged in descending order
of LacZ activation. As evident from the second column in
Table II,
-galactosidase levels varied from 7- to 70-fold over the
background levels in
swi6 parental strain MY10Z.
|
To assess whether the mutations were recessive, each mutant was mated
to swi6 yeast strain MY2, and the resulting diploids assayed for
-galactosidase activity. As shown in the third column of
Table II,
-galactosidase activity was extinguished in all cases when
mutants were mated to nonmutant cells, indicating that all 30 MCB-activating mutations were recessive. When the diploid cells were
sporulated, and inheritance of the MCB-activating phenotype was
determined by random spore analysis, 50% of the spores showed the
mutant phenotype, consistent with the segregation pattern expected for
a single mutated gene.
To begin to assign the mutations to complementation groups, a MAT
derivative of strain MY74, obtained by sporulating a MY74 × MY2
diploid, was mated to all 30 of the original mutants and the resulting
diploids assayed for
-galactosidase. As shown in the fourth column
of Table II, all of the diploids showed high
-galactosidase
activity. The failure of any diploids to show the low
-galactosidase
activity characteristic of nonmutant cells indicated that all 30 mutations were in the same complementation group. In the fifth column
of Table II, each mutant allele is identified by a number indicating
its relative placement in the array of phenotypes. The various alleles
gave varying degrees of MCB reporter gene activation, suggesting that
at least the more poorly activating mutations were not null alleles.
Although they restored MCB reporter gene expression, the mutations did not suppress other aspects of the swi6 phenotype. None of
the mutations listed in Table II corrected the abnormal morphology of
swi6 cells, and at least the 1-21 mutation
(the only one tested) did not suppress the synthetic lethality of
swi6 swi4 double mutations (data not shown).
Strains with the more strongly activating mutations grew significantly slower than parental cells. Strain MY90 grew with a generation time nearly equivalent to nonmutant parental cells. Therefore MY90 and strains derived from MY90 that carried the 1-21 allele were used in further characterization of the MCB-activating mutation.
The wild type allele of the gene identified by the mutations was cloned
by complementation. Strain MY43Z, a derivative of MY90 that carried the
1-21 mutation but lacked the integrated LEU2-marked MCB/HIS3 reporter gene, was
transformed with the high copy, 2µ-based libraries YL1, -2, and -3 (19) or with the single copy, ARS/CEN-based
library YpH1. Transformants were selected using the LEU2
marker on the library vector and were then screened for white color in
filter -galactosidase assays. Of 70,000 high copy transformants
screened, 200 were white. Of these, only 16 were white when replated
and reassayed by filter
-galactosidase assay. When the 16 were
assayed by liquid
-galactosidase assay, only three showed complete
suppression of reporter gene activity. In a similar screen of 20,000 single copy transformants, only one transformant showed complete
suppression of reporter gene activity. Plasmids from the four
transformants showing complete reporter gene suppression were isolated
in Escherichia coli and reshuttled to MY43Z cells to confirm
that they complemented the mutation. All four plasmids restored
-galactosidase activity to the low level expected for a
swi6 cell.
Restriction mapping and partial sequencing of the four plasmids established that all contained permutations of the same locus near TRP4 on chromosome IV. Only two open reading frames were common to all four plasmids and were thus candidates for encoding the complementing activity. One open reading frame (residues 9743-10697 of cosmid clone YSCL9476, GenBankTM accession no. [GenBank]) predicted a protein with the seven hydrophobic domains characteristic of serpentine membrane proteins. The second open reading frame predicted a 317-amino acid protein that was 62% identical to E. coli thioredoxin reductase. While we were carrying out our experiments, Chae et al. (27) reported the cloning and sequencing of this thioredoxin reductase gene and named it TRR1 (EMBL accession no. [GenBank]). Our sequence varies from the deposited sequence at two residues, which predict Thr for Ala amino acid substitutions at residues 100 and 110. A protein 82% identical to Trr1 is predicted by open reading frame YHR106w on chromosome VIII (GenBankTM accession no. [GenBank]), which we tentatively named Trr2.
To test which open reading frame had the complementing activity, a
2.1-kb Bgl2/XhoI fragment containing only the
serpentine protein gene, or a 2.6-kb EcoRI fragment
containing only the TRR1 gene was subcloned into YEp181 and
transformed into MY43Z yeast. Only in yeast transformed with the latter
subclone was -galactosidase activity extinguished, proving that
TRR1 possessed the complementing activity.
To establish that TRR1 and the gene identified in the mutant
screen were allelic, the chromosomal TRR1 locus was
non-disruptionally tagged in a nonmutant swi6 strain by
insertion of a LEU2-marked plasmid. Integration at the
TRR1 locus was confirmed by Southern blot analysis. The
TRR1:LEU2-tagged strain was mated to mutant strain MY43 and
inheritance of the Leu+ and
-galactosidase+
phenotype was monitored. In 16 dissected tetrads and 43 random spores,
the Leu+ and
-galactosidase+ phenotype were
always inherited reciprocally, thus establishing that TRR1
and the mutation responsible for LacZ reporter gene activation were allelic.
To determine the effect of disrupting TRR1 on cell viability
and on MCB reporter gene expression, the TRR1 coding region
was transplaced by HIS3 in a diploid strain. Disruption of
the TRR1 gene was confirmed by Southern blot analysis. When
the heterozygous trr1:HIS3 deletion mutant
(MY196) was sporulated and asci were dissected, most tetrads yielded
only two or three colonies, two of which were large and
His
. In those tetrads that gave four colonies, two were
large and His
and two were small and His+. We
concluded that disruption of TRR1 resulted in poor
viability, and that in those
trr1:HIS3
disruptants that managed to survive, the growth rate was significantly
slower than in TRR1 cells. The same pattern was obtained
when
trr1:HIS3 segregants were backcrossed several times to W303-1. Thus, poor viability and slow growth was
intrinsic to the disruption of TRR1 and not due to the
segregation of other potential polymorphisms in the diploid used for
the gene disruption. The viability problem was even more evident when
spores were plated on supplemented minimal plates. When random spores were directly plated on supplemented minimal plates, no
trr:HIS3 spores formed colonies. When the experiment was
repeated but spores were allowed to form colonies on YEPD plates and
then were replica-plated to supplemented minimal plates, some small
trr1:HIS3 colonies were evident, but at much lower
frequency than the 50% expected. The results suggested that
trr1:HIS3 mutants have trouble germinating on YEPD plates
and cannot germinate at all on supplemented minimal medium.
When a dissected tetrad that yielded four viable spores was analyzed to
quantitate the effect of deleting TRR1 on cell growth rate,
the trr1:HIS3 segregants grew with an average
doubling time of 3.1 h, which was 70% longer than the 1.8-h
doubling time of TRR1 segregants.
The viability of trr1 null mutants allowed us to test the
effect of TRR1 disruption on MCB reporter gene activity. A
trr1:HIS disruptant (MY199) was mated to
swi6:TRP1 strain MY2Z, and segregants with the
four expected haplotypes were isolated. As shown in Table III,
trr1:HIS3
swi6:TRP1 segregants showed 50-fold higher
-galactosidase levels than their TRR1
swi6
counterparts. An effect of deleting TRR1 was also observed
in SWI6 cells, where
trr1:HIS3
segregants showed 2.5-fold higher
-galactosidase activity than their
TRR1 counterparts. In
swi6 cells, the 50-fold
effect of the
trr1 deletion mutation on MCB reporter gene
expression was roughly equivalent to that of the strongest activating
trr1 alleles isolated in the mutant screen (Table II).
|
As loss of function trr1 mutations restored MCB reporter
gene activity in swi6 yeast, it suggested that Trr1
protein functions as an inhibitor of MCB element activity. We therefore
investigated whether introduction of the TRR1 gene on a high
copy plasmid suppressed MCB reporter gene activity in
SWI6+ yeast. It did not. Wild type yeast
transformed with a high copy TRR1 plasmid showed the same
level of reporter gene activity as wild type yeast transformed with a
control plasmid (data not shown). We concluded that a single normal
copy of the TRR1 gene produced sufficient Trr1 protein to
maximally suppress reporter gene activity.
Using
genetic screens similar to ours, a number of global repressors of
transcription have been isolated in yeast (28). A characteristic of
global transcriptional repressors is that recessive mutations in the
encoding gene result in elevated expression from UAS-less basal
promoters such as the 178CYC1 promoter. To determine
whether trr1 mutations enhanced transcription from a CYC1 promoter lacking a functional UAS, the effect of the
trr1-21 mutation on expression of a UAS-less
178CYC1/LacZ gene or a mutant MCB/LacZ gene, in which the upstream MCB elements were
mutated to ACtaGT, was determined. In addition, the effect of the
trr1-21 mutation on the expression of a
312CYC1/LacZ reporter gene carrying the native
CYC1 UAS and on the expression of a SCB/LacZ
reporter gene (pBd1390) (29) carrying three upstream SCB elements
(consensus CACGCAAAA) derived from the HO gene was
determined.
As shown in the top rows of Table IV, the
trr1-21 mutation elevated -galactosidase activity
12-25-fold in cells carrying either the MCB/LacZ or
SCB/LacZ reporter gene. In contrast, the trr1-21
mutation elevated
-galactosidase activity only 2-4-fold in cells
carrying the
312CYC1/LacZ,
178CYC1/LacZ, or mutMCB/LacZ reporter gene.
Strains BY600 and MY43 have an integrated ho:LacZ gene.
Although this gene is silent in
swi6 yeast (2), it was possible that the gene was activated in trr1-21 yeast and
was contributing to observed
-galactosidase activity. To control for
this possibility,
-galactosidase levels were also assayed in cells
transformed with a vector (YEp195) lacking any LacZ reporter gene. As shown in the right column of Table IV,
-galactosidase activity in YEp195 transformants was low and unchanging, which indicated that the trr1-21 mutation had no effect on
expression of the integrated ho:LacZ gene.
|
The specificity of TRR1 involvement in MCB activation was
also tested in trr1 null mutants. As shown in the lower
part of Table IV, the
trr1:HIS3 mutation
elevated
-galactosidase 17- to 20-fold in cells carrying the
MCB/LacZ or SCB/LacZ reporter gene but only
3-fold in cells carrying the mutMCB/LacZ,
178/LacZ or
312/LacZ reporter genes. In
summary, the data in Table IV indicated that trr1 mutations
weakly and nonspecifically activated all reporter genes utilizing the
basal CYC1 promoter. However, the trr1 mutations
gave an additional, specific, 5-fold activation of reporter gene
expression if the basal CYC1 promoter was provided with
upstream MCB or SCB elements.
Having established that trr1 mutations elevated
MCB reporter gene activity, we next investigated whether the mutations
affected expression of endogenous MCB-containing genes. The
TMP1 gene encoding thymidylate synthase is expressed
maximally at G1/S and has the best characterized MCB
element region (3). TMP1 mRNA was not detectable by the
Northern blot method, so a RNase protection assay was used to measure
TMP1 mRNA in cells with or without trr1 mutations (data not shown). Inclusion of a standard curve generated using synthetic TMP1 pseudo mRNA allowed absolute
quantitation of TMP1 mRNA levels. Assuming 1 pg of total
RNA/yeast cell, 5 × 107 exponentially growing wild
type yeast (strain MY224) or swi6 yeast (strains MY1, 10, 216, and 217) yielded about 500 amol of TMP1 mRNA, which
was equivalent to about 10 copies of TMP1 message/cell. TMP1 mRNA levels were unchanged in several
trr1 mutants selected in the mutant screen (strains MY100,
68, 95, 90, 61, and 62) and in several
trr1 deletion
mutants (strains MY210, 213, and 214). Northern blots assays were used
to determine the activity of three other G1/S-specific,
MCB-containing genes: RNR1, CDC9, and
SWI4. As with TMP1, no significant change in the
level of expression of these genes was associated with the
trr1 mutations (data not shown). The trr1
mutations similarly did not affect the level of expression of five
nonperiodic genes: SWI6, BRY1(SKN7),
LEU2, URA3, and P1, or the S
phase-specific H2A gene (data not shown). Thus, at least as measured in
asynchronously growing cells, trr1 mutations that strongly
and specifically activated MCB-containing reporter genes did not
activate several MCB-containing endogenous genes.
Although the trr1 mutations did not affect endogenous MCB
gene mRNA levels, neither did deletion of SWI6, a gene
known to encode an important MCB element regulator. Previous studies
have also reported little effect of SWI6 deletion on
asynchronous cell CDC9, RNR1, and TMP1
mRNA levels (8, 9). However, in these previous studies, deletion of
SWI6 did disrupt the normal periodicity of these
transcripts. We therefore investigated whether the trr1 mutations affected endogenous MCB gene periodicity. Strains carrying the trr1-21 allele were selected for study because they
grew at wild type rates. (Strains carrying the more strongly activating alleles listed in Table II or strains with the trr1
disruption mutation grew slowly, and therefore, perturbations in
expression patterns would be more difficult to interpret.) As
swi6 cells were difficult to synchronize and were already
known to show altered DNA synthesis gene periodicity (8, 9), we
investigated the effect of trr1-21 on DNA synthesis gene
periodicity in SWI6 cells. Cells were synchronized using
centrifugal elutriation (4). To improve the size homogeneity of the
population, diploid cells were used. Cells that were either
trr1-21/trr1-21 homozygotes or TRR1/trr1-21
heterozygotes were analyzed. To facilitate measurement of
TMP1 mRNA, both cell types carried the high copy
TMP1-encoding plasmid pEM54. Synchrony was monitored by
determining the budding index.
As shown in Fig. 1A, bud emergence in both
populations began about 60 min after elutriation. Northern blot
analysis of RNA from the synchronized cells (Fig. 1B) showed
that mRNA levels expressed from three different MCB genes
(TMP1, RNR1, and SWI4) remained
periodic in the trr1-21 mutant. However, in contrast to the
simultaneous onset of budding, peak MCB gene mRNA levels in
trr1-21 cells occurred about 15 min earlier than in
TRR1 cells. Fig. 1C shows TMP1
mRNA levels, normalized to the nonperiodic LEU2 message,
and RNR1 and SWI4 mRNA levels, normalized to
the nonperiodic SWI6 message.
The effect of trr1 mutations on MCB gene periodicity was
also investigated in cells synchronized by release from a
cdc15 block. Fig. 2 shows RNR1
mRNA in cdc15-synchronized trr1-21 or wild
type yeast. In both populations bud emergence began 48 min after
release from the nonpermissive temperature. As was previously observed for elutrially synchronized yeast, RNR1 mRNA remained
periodic in cdc15-synchronized trr1-21
yeast.
We also investigated whether introduction of TRR1 on a high copy plasmid affected endogenous MCB gene periodicity. It did not. The G1/S-specific pattern of RNR1 expression was remarkably similar in cells transformed with a high copy TRR1 plasmid and cells transformed with a control plasmid. The results suggested that overproduction of thioredoxin reductase did not alter MCB gene periodicity.
Mechanism of trr1 Effect on MCB Element ActivityDespite its
name, the enzyme thioredoxin reductase may reduce substrates other than
thioredoxin. For example, in E. coli, mutations in the
TrxB gene encoding thioredoxin reductase, but not the
TrxA gene encoding thioredoxin, allow the accumulation of
active alkaline phosphatase in the cytosol (30), suggesting that
thioredoxin reductase can influence the structure of certain cytosolic
proteins by a thioredoxin-independent mechanism. To determine whether
trr1 mutations in yeast activated MCB element activity by a
thioredoxin-dependent mechanism, MCB reporter gene activity
was determined in swi6 strains lacking one or both of the
S. cerevisiae thioredoxin genes TRX1 and
TRX2. As shown in Table V,
swi6
yeast in which both thioredoxin genes were deleted showed 50-fold
higher
-galactosidase activity than
swi6 yeast in
which the thioredoxin genes were intact. Table V also shows that
TRX1 and TRX2 were not equivalent in their
ability to repress MCB reporter gene activity. Strains in which only
TRX2 was deleted showed the same low
-galactosidase
activity as
swi6 cells. In contrast, strains in which
only TRX1 was deleted showed substantially higher
-galactosidase activity, although not nearly as high an activity as
in cells in which both thioredoxin genes were deleted. The results in
Table V show that deleting both thioredoxin genes gave about the same
level of MCB/LacZ reporter gene activity as deleting
TRR1. Thus, we concluded that the negative effect of thioredoxin reductase on MCB element activity was mediated through thioredoxin.
|
MCB-controlled genes are activated shortly after START and inactivated
in mid-S phase (3, 4). Thus, MCB reporter gene activation in
trr1 mutants could be a consequence of disproportional expansion of a cell cycle compartment during which MCB elements are
active. For example, if trr1 mutations delayed cytokinesis by prolonging S or G2, daughter cells would be larger and
pass through early G1 more quickly. The result would be
that more cells would be in the S/G2 phases of the cell
cycle than in G1 phase. To investigate the possibility that
trr1 mutations disproportionately affected the duration of
cell cycle compartments, exponentially growing TRR1 wild
type and trr1-21 mutant cells were stained with propidium
iodide and analyzed by flow cytometry. As shown in Fig. 3A, in both a SWI6 background
(compare top two panels) or swi6 background
(compare bottom two panels), the DNA profiles of
TRR1 wild type and trr1 mutant cells were very
similar, indicating that the trr1-21 mutation did not
disproportionately expand the S/G2 phases of the cell
cycle. In contrast to the lack of effect of trr1 on the
frequency of G1 and S/G2 cells, in both
TRR1 and trr1-21 cells, deletion of
SWI6 increased the percentage of G2/S cells at
the expense of G1 cells (compare top two panels
with bottom two panels of Fig. 3A).
If trr1 mutations delayed cytokinesis without affecting mass
accumulation, trr1 cells should be larger than
TRR1 cells. Forward angle light scattering (FALS) is a
measure of the cross-sectional area of an object as it passes the flow
cytometer interrogation point. FALS values thus were used to assess any
effect of trr1-21 on cell size. As shown in Fig.
3B, the trr1-21 mutation did not affect the
modal FALS value of either SWI6 cells (compare top two
panels) or swi6 cells (compare bottom two
panels), indicating that trr1-21 did not increase
average cell size. As expected, deletion of SWI6 did
increase the modal FALS value (compare top two panels with
bottom two panels), consistent with the microscopic observation that
swi6 cells are larger than
SWI6 cells (2).
Based on DNA content and FALS data, the trr1-21 allele did
not alter the relative distribution of cells within the cell cycle and
did not increase cell size. The trr1-21 mutant was
initially selected for characterization because it had a doubling time
that was only slightly longer than TRR1 parental cells (2.5 h for MY90 versus 2.2 h for MY10). Other
trr1 alleles, that more strongly activated MCB reporter gene
expression, resulted in significantly longer doubling times. However,
flow cytometric analysis showed that even the most strongly activating
trr1 alleles listed in Table II, as well as the
trr1 null mutation, caused no shift in the relative
proportion of G1 and S/G2 cells (data not
shown). We concluded that even in the more slowly growing
trr1 mutants, which showed high levels of MCB reporter gene
activation, all phases of the cell cycle were expanded equally.
As the thioredoxin reductase/thioredoxin system presumably functions to
balance oxidative processes in the cell, we investigated whether
trr1 mutations resulted in hypersensitvity to the oxidant H2O2. To assess H2O2
sensitivity, exponentially growing cells were spread on YEPD plates,
and a drop of H2O2 was introduced at the center
of the plate. After allowing cells to grow for 12-24 h, the diameter
of the zone in which growth was suppressed, the halo, was determined.
In all yeast strains tested in which the TRR1 gene was
intact, halos 3.5-4.5 centimeters in diameter were obtained. In all
strains tested in which the TRR1 gene was deleted or
mutated, halos 5.5-6.5 centimeters were obtained. An example of the
difference in halo diameter between trr1 mutants and wild type cells is shown in Fig. 4. The greater diameter of
the halos in trr1 mutants was consistent with the idea that
the mutations resulted in greater sensitivity to oxidizing
conditions.
The H2O2 halo assays provided further insight
into the mechanism by which trr1 mutations activated MCBs.
As yeast possess a thioredoxin-dependent peroxidase (27),
exposure to H2O2 might be expected to oxidize
thioredoxin and allow MCB reporter gene expression in
swi6 yeast. To test whether H2O2
allowed MCB reporter gene activation in
swi6 yeast, cells
that had been grown in H2O2 halo assays were
replica-plated to filters and assayed for
-galactosidase activity.
Results of such an assay are shown in Fig. 4. The left-hand panels show control cells not exposed to
H2O2. As expected,
-galactosidase activity
was high in
swi6
trr1 cells (strong
blue color) and low in
swi6 cells. The
right-hand panels show cells exposed to H2O2. Significantly, in
swi6
cells, a ring of high
-galactosidase activity was observed in the
region where cells were presumably exposed to the highest non-lethal
concentration of H2O2. Induction of the
MCB/LacZ reporter gene by H2O2 is
consistent with the idea that oxidation of thioredoxin results in MCB
element activation.
Although trr1 mutations activated the
MCB/HIS3 reporter and MCB/LacZ reporter genes,
they did not noticeably affect endogenous MCB gene mRNA levels. The
lack of effect of trr1 mutations on TMP1,
RNR1, and SWI4 mRNA levels is reminiscent of
the disparate effect of deleting SWI6 on reporter gene and
endogenous gene expression. Deletion of SWI6 strongly
represses 178CYC1/LacZ reporter genes that are
dependent on either synthetic MCB element clusters (8) or on natural
MCB elements as they are found in the context of a 55-base pair
fragment of the TMP1 upstream region (9). In contrast,
deletion of SWI6 has little effect on the levels of several
mRNAs encoded by endogenous MCB-containing genes such as
TMP1, CDC9, POL1, RNR1,
SWI4, CLN1 and CLN2 (8, 9). Foster et al. (7) showed a small but significant effect of deleting SWI6 on SWI4 mRNA levels. Despite the lack of
a strong effect of deleting the trans-acting factor Swi6, in
cases where it has been examined, mutation of the cis-acting
MCB sites have a strong negative effect on endogenous gene mRNA
levels (3, 7). To summarize, cis-acting mutations that
destroy MCB sites have strong effects on both endogenous and reporter
gene expression, whereas trans-acting mutations such as
deletion of SWI6 or mutation of TRR1 have little
effect on endogenous gene expression but strong effects on reporter
gene expression.
One plausible model to explain the disparity is that in the absence of
Swi6 protein, MCB elements are targets for both inhibitory and
stimulatory mechanisms affecting transcription. In the context of most
native promoters, MCB elements may be targeted primarily by the
positive-acting mechanism, which results in a moderate level of
constitutive transcription. In the context of the heterologous CYC1 promoter, MCB elements may be targeted primarily by the
negative-acting mechanism, which results in a low level of
transcription. If the negative-acting mechanism requires thioredoxin
reductase for its activity, it would explain why trr1
mutations activate MCB reporter genes in swi6 yeast.
The above explanation raises the question of whether thioredoxin reductase normally has any role in the regulation of endogenous MCB gene expression. At present, we do not know the answer. Neither deletion nor overexpression of TRR1 affected the level of expression of any of the endogenous MCB genes monitored. However, Muller (31) has shown that deletion of both yeast thioredoxin genes results in increased RNR1 and RNR2 mRNA levels. We were not able to duplicate this result using identical strains and similar conditions, raising the possibility that some aspect of the way in which we grow yeast or analyze mRNA may obscure an effect of thioredoxin or thioredoxin reductase gene mutations on MCB gene mRNA levels. Furthermore, other endogenous MCB genes, outside the subset analyzed in our study, may behave more like the MCB reporter genes in terms of Swi6-dependence and Trr1-sensitivity.
How might trr1 mutations activate MCB elements? Thioredoxin
reductase regenerates reduced thioredoxin from oxidized thioredoxin using NADPH as electron donor. Diminished levels of reduced thioredoxin in trr1 mutants could lead to oxidation of regulatory thiols
in proteins that either directly or indirectly control MCB element activity. Oxidation may either activate a positive-acting control protein, or inactivate a negative-acting control protein. Redox control
of transcription factor activity has been suggested for NFB (32),
Fos/Jun (33), glucocorticoid receptor (34), and the MyoD-interacting
protein E2A (35). In these vertebrate examples, protein oxidation is
correlated with loss of DNA binding or transcriptional activity.
However, in bacteria, oxidation of the OxyR regulatory protein is
associated with enhanced transcriptional activity (36). A direct role
for thioredoxin in redox control of transcription has been suggested
for NF
B (32). An indirect role for thioredoxin has been suggested
for Fos/Jun regulation, where the proximal redox effector protein is
thought to be Ref1 (33). Recently, in S. pombe, a mutation
in a gene encoding thioredoxin reductase was shown to circumvent the
cell cycle arrrest induced by human p53 (37). Outside the realm of
transcription factors per se, thioredoxin has been
implicated in the folding or conformational regulation of several
eucaryotic and prokaryotic proteins (reviewed by Buchanan et
al. (38)).
In addition to its activity as a protein disulfide reductase,
thioredoxin is the proximal donor of electrons during reduction of
ribonucleoside diphosphates to deoxyribonucleoside diphosphates by
ribonucleotide reductase, during reduction of sulfate to sulfite by
adenosine 3-phosphate 5
-phosphosulfate reductase, and during reduction of H2O2 to H2O by
thioredoxin-dependent peroxidase (27).
In light of the activities of thioredoxin, one intriguing model for thioredoxin involvement in G1/S transcriptional regulation involves ribonucleotide reductase. According to the model, MCB genes may be subject to negative control by a thioredoxin-dependent regulatory protein. In non-G1/S cells the regulatory protein would be maintained in a reduced state by an adequate supply of reduced thioredoxin. However, after replication origins are triggered at G1/S, the cell deoxynucleoside triphosphate (dNTP) pools would be quickly consumed through incorporation into DNA. Muller (31) estimated the dNTP pools in budding yeast to be only 5% of the 6 × 107 bases minimally needed to replicate the genome. Freed from dNTP feedback inhibition, ribonucleotide reductase would begin to rapidly convert ribonucleotides to deoxyribonucleotides, quickly depleting the pool of reduced thioredoxin. Proteins with thioredoxin-dependent thiols would become oxidized, triggering conformational changes that either directly or indirectly activate transcription of MCB/SCB-dependent genes. As proteins involved in DNA precursor synthesis accumulate and the dNTP demand becomes satisfied, reduced thioredoxin would begin to reaccumulate, and the thioredoxin-sensitive transcription system would be returned to an off state. In considering how accumulation of DNA precursor synthesizing enzymes could satisfy the dNTP demand, it is noteworthy that the TRR1 gene itself contains an upstream MCB element and is maximally expressed at G1/S.2
As an initial test of the idea that an episode of RNR-mediated
thioredoxin oxidation at G1/S may contribute to MCB gene
induction, cdc15-synchronized cells were incubated in the
presence or absence of the RNR inhibitor hydroxyurea (HU), and
endogenous MCB gene mRNA levels were measured by Northern blot
analysis. Both the nontreated and HU-treated populations began to bud
about 45 min after release from the nonpermissive temperature,
indicating that HU did not block START or replication-independent
processes downstream from START. Coincident with budding, nontreated
cells showed a severalfold increase in RNR1, SWI4
and TRR1 mRNA. The increase in MCB gene mRNA was
followed by an increase in H2A message, which indicated that
the nontreated cells had crossed the G1/S border by 60 min
and were actively replicating DNA. In contrast, most of the increase in
RNR1 mRNA and all of the increase in SWI4 and TRR1 mRNA did not occur when
cdc15-synchronized cells were incubated in HU. The increase
in H2A mRNA also did not occur in the HU-treated population, consistent with the idea that DNA replication was blocked
in the absence of DNA precursor biosynthesis. Similar effects of HU on
MCB gene induction were observed in -factor-synchronized cells, as
long as the drug was added several minutes before release from the
pheromone block. The experiments with HU did not distinguish whether
suppression was due to inhibition of ribonucleotide reductase per
se or was due to inhibition of replication. It is difficult to
distinguish experimentally between an effect on RNR activity and an
effect on replication. Drugs or mutations that block replication would
also be expected to inhibit RNR activity due to dNTP feedback inhibition, and drugs that inhibit RNR would also be expected to block
replication due to exhaustion of dNTP pools. In either case, our
results showed that MCB gene induction, unlike other post-START events
such as bud emergence, was inhibited by hydroxyurea.
Critical testing of the model that RNR-mediated oxidation of thioredoxin contributes to MCB gene induction at G1/S will require the development of an assay capable of measuring the REDOX state of thioredoxin during the cell cycle. One attractive feature of the model is that it suggests a biochemical mechanism for linking the onset of DNA replication to induction of specific gene transcription at G1/S.
We thank Linda Breeden, Eric Muller, and Alan Bakalinsky for reviewing the manuscript and suggesting improvements; Anja Bauman and Timothy Miller for assistance in assigning mutants to complementation groups; Reg McParland for DNA sequencing; Corwin Willard, David Barnes, and the OSU Environmental Health Sciences Center (NIEHS Center Grant ES00210) for providing the expertise and instrumentation required for flow cytometry and elutrial synchronization; and Linda Breeden, Eric Muller, Evan MacIntosh, Leland Johnston, Paul Bartel, Stanley Fields, and Steven Elledge for providing plasmids and yeast strains.