From the Institute of Molecular Biology and Biotechnology-Foundation of Research and Technology, Vassilika Vouton, P. O. Box 711 10 Heraklion, Crete, Greece
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ABSTRACT |
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Cyc8(Ssn6)-Tup1, a general co-repressor complex,
is recruited to promoter DNA via interactions with DNA-binding
regulatory proteins and inhibits the transcription of many different
yeast genes. Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1
protein, and requires specific components of the RNA polymerase II
holoenzyme such as Sin4 and Rgr1. In this study we test the transcriptional activity of the Cyc8 subunit using a lexA
operator-containing reporter. We show that a LexA-Cyc8
hybrid stimulates transcription when expressed in a
tup1 An important class of pleiotropic transcriptional regulators
includes intermediary proteins such as co-activators and co-repressors. These protein factors are tethered to specific promoters mainly by
contacting DNA-binding factors and regulate transcription either by
interacting with components of the Pol II holoenzyme or by modifying
chromatin structure, or both (1, 2). The human co-activator CBP/p300
(3), the yeast SAGA complex (4), and the human nuclear receptor
co-repressors SMRT and N-CoR (5) are among the best characterized
examples of this growing protein family. Interestingly, some of these
factors have dual function on specific promoters; for example CBP/p300,
which mediates activation of interferon In the yeast Saccharomyces cerevisiae, two physically
associated proteins, Cyc8(Ssn6) and Tup1, inhibit the transcription of
many diversely regulated genes when their expression is not required
(6-8). It is well established that Cyc8-Tup1 acts as a co-repressor
complex that does not bind DNA directly but is recruited to different
promoters via interactions with specific DNA-binding regulatory
proteins. The repression function of the complex is performed by a
specific domain of Tup1 (8). When the Tup1 repression domain is brought
upstream of an active test promoter through the DNA binding domain of
LexA, it inhibits transcription independently of Cyc8. Moreover, this
domain is required for repression of natural genes such as glucose,
oxygen, and cell-type regulated genes. It has been postulated that
multiple mechanisms are responsible for Tup1 repression. Tup1 interacts
with histones H3 and H4 and may position nucleosomes over the
transcription start point, suggesting that Tup1 might repress
transcription by modifying chromatin structure (9, 10). However,
evidence from other studies argue that Tup1 inhibits the function of
the basic transcription machinery; Tup1 repression was reconstituted in
an in vitro transcription system in the absence of chromatin
(11), and mutations in specific components of the RNA polymerase II
holoenzyme complex, such as the mediator proteins Sin4, Rgr1, Srb10,
and Srb11, weaken the Tup1 repression activity (12-15).
Previous studies suggested that Cyc8 does not directly inhibit
transcription but contacts specific DNA-binding regulatory proteins (8,
18). The N-terminal region of Cyc8 consists of 10 tandem repeats of a
sequence motif termed tetratrico peptide repeat
(TPR)1 (16). TPRs serve as
protein-protein interaction domains, and more importantly in the case
of Cyc8, TPRs exhibit distinct interaction specificity although they
are similar in primary structure (17-20). TPR1, TPR2, and TPR3 contact
Tup1, while different combinations of TPR4 to TPR10 mediate recruitment
of Cyc8-Tup1 to different promoters (18). Based on these observations
it was proposed that the function of Cyc8 is to link Tup1 to distinct,
structurally dissimilar, DNA-bound repressor proteins. Consistently
with this linker function, derivatives of Cyc8 that contain only the
TPR domain are sufficient for repression. On the other hand, the
C-terminal domain which comprises more than half of the protein appears
to be dispensable (16-18).
Recent genetic data suggested that Cyc8-Tup1 might also play a positive
role in transcriptional control. More specifically, activation of the
CYC1 gene transcription by the Hap1 transactivator and
maximal induction of SUC2 gene both require functional Cyc8 protein (26, 27). In this report, we present direct evidence that
Cyc8-Tup1 indeed plays diverse roles in transcriptional regulation. Expression of Cyc8-Tup1 repressible genes is stimulated in response to
specific signals, and under these conditions, the fate of the Cyc8-Tup1
co-repressor has been unclear for most of the cases. Our data suggest
that this complex can convert to a co-activator of CIT2, a
gene encoding a peroxisomal isoform of citrate synthase that is
expressed upon inducing conditions of mitochondrial dysfunction (21).
We show that Cyc8-Tup1 is tethered to the CIT2 promoter by
interacting with Rtg3, a DNA-binding transactivator of CIT2. Genetic analysis indicates that basal (uninduced) expression of CIT2 is inhibited by Tup1, but its transcriptional
activation is mediated by the second component of the complex, the Cyc8
subunit and specifically requires the C-terminal domain of this protein.
The transcriptional activity of Cyc8 was further examined using a
synthetic reporter promoter and a LexA-Cyc8 hybrid. Previous studies
have shown that LexA-Cyc8 represses transcription by recruiting the
Tup1 repressor (18). Here we show that LexA-Cyc8 can activate transcription when Tup1 is absent or when the Tup1 repression is
substantially impaired, as it is in a sin4 Yeast Strains, Media, and Growth Conditions--
All strains are
derivatives of FT5 strain (MAT
Standard synthetic media were used; YPD- and YPR-rich media contained
2% glucose or 2% raffinose, respectively. CS minimal media were
supplemented with 0.6% casamino acids and glucose or raffinose as the
carbon source. Mitochondrial dysfunction was caused by prolonged
treatment of cells (~48 h) with 20 µg/ml ethidium bromide in YPD
medium. CIT2 induction was monitored in exponential cultures
growing in YPR medium. Standard procedure was used for routine yeast
transformations, while for high efficiency yeast transformation the
TRAFO protocol was followed (22).
Plasmid Constructs--
All Cyc8, Tup1, and LexA derivatives
expressed from the YCp91 vector and have been described previously
(18). Briefly, the centromeric vector YCp91 (TRP1 marked)
contains the ADH1 promoter and 5'-untranslated sequence
(including the ATG start codon), followed by the SV40 nuclear
localization signal and the HA1 epitope from the influenza virus (flu
epitope), a polylinker sequence, and the CYC8 termination region (8).
The reporter plasmid JK103 (23) is a URA3 marked multicopy
plasmid that expresses the LacZ reporter gene from a minimal
promoter consisting of four overlapping LexA-binding sites upstream of
the GAL1 TATA box. The Gal4 activation domain-genomic
library (used for the two-hybrid screening) was constructed in the
pACT2 vector.2 pACT2, a 2µ,
LEU2 marked vector contains the ADH1 promoter and transcription start site followed by sequences consisting of nuclear localization signal, the activation domain of Gal4, and polylinker sequence (in which random genomic fragments have been inserted) ending
to the termination region of the ADH1 gene.
Two-hybrid Screening for Cyc8-Tup1 Interacting
Proteins--
LexA-Cyc8, cloned in the YCp91 expression vector, was
used to transform L9FT5 along with the LacZ reporter plasmid JK103
(23). L9FT5 yeast transformants appear white on X-Gal indicator plates and are sensitive for growth in low concentrations of 3-aminotriazole (0.5-1.0 mM), a competitive inhibitor of His3
enzymatic activity. The pACT2 library was used to transform this
strain, and cells were recovered by shaking in selective liquid medium
(SC, containing 2% glucose and 2% galactose) for 10 h at
30 °C. Five million independent transformants were scored for growth
in minimal media containing 3-aminotriazole at a concentration of 20 mM. 200 colonies, scored as positives, were selected
(normal growth in 20 mM 3-aminotriazole), but less than
half of them (~96) appeared blue on X-Gal plates. Positive
transformants containing pACT2-derived plasmids were rescued in the
Escherichia coli KC8 strain (constructed by K. Struhl) based
on the ability of the yeast LEU2 gene (pACT2 is a
LEU2 marked plasmid) to complement the respective E. coli auxotrophy. 39 of 96 plasmids analyzed reproducibly supported
3-aminotriazole resistance and high GST Interaction Assay--
A BamHI-PvuII
fragment containing Rtg3-68 was cloned in the T7 expression vector
pRSETC and was used to direct coupled transcription translation
(Promega T7 TNT). 35S-Labeled Rtg3 protein (10 µl) was incubated with approximately 2 µg of agarose
bead-immobilized GST-Cyc8 protein (18) in a volume of 100 µl
containing 20 mM Tris-acetate, pH 7.4, 10% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.15 M potassium acetate, and 1× complete protease inhibitors
(Boehringer) for 2 h at 4 °C. Following incubation the beads
were extensively washed in the above buffer, eluted in SDS-PAGE gel
loading buffer, and analyzed by SDS-PAGE.
EMSA--
Whole cell protein extracts from wild type and
cyc8 RNA Analysis--
Total cellular RNA was extracted from yeast
cells grown in the appropriate medium, using the acid phenol method
(25), and was fractionated in 1.4% agarose gels containing 5.5%
formaldehyde. RNA was transferred to nylon membrane and hybridized with
32P-labeled probes generated by nick translation. For
CIT2 and TBP probes, polymerase chain reaction fragments
containing the entire coding sequence (from ATG to termination codon)
of the respective genes were used.
LacZ Assays--
Transcriptional Activation by Cyc8--
Recent genetic evidence
suggested that Cyc8-Tup1 might play a positive role in transcriptional
regulation. Mutations in the CYC8 gene adversely affect both
Hap1-mediated stimulation of CYC1 and maximal induction of
SUC2 transcription (26, 27). Based on these observations, we
directly tested whether Cyc8 can stimulate transcription by analyzing
the activity of a LexA-Cyc8 hybrid protein on a
GAL1-LacZ synthetic reporter that contains a LexA operator upstream of the TATA element. Wild type and isogenic tup The Activation Domain of Rtg3 Associates with the Cyc8-Tup1 Protein
Complex--
If Cyc8-Tup1 acts as a co-activator of certain natural
promoters, then it would be expected to directly interact with
promoter-specific DNA-binding activator protein(s). We explored this
possibility by seeking such Cyc8-Tup1 interacting proteins (activator
proteins), using a yeast two-hybrid screen (see "Experimental
Procedures"). We generated a strain that expresses HIS3
and LacZ reporter genes under the control of promoters
containing a LexA-binding site. This strain was co-transformed with a
LexA-Cyc8 expressing plasmid along with a library of random yeast
genomic fragments fused to the Gal4 activation domain. Library plasmids
supporting high levels of both HIS3 and LacZ
reporter genes were recovered and sequenced. This selection scheme
revealed 39 positive clones encoding regions from seven different
proteins. Consistent with the co-repression function of Cyc8-Tup1, most
of these were DNA-binding repressor proteins that inhibit transcription
in a Cyc8-Tup1-dependent
manner.3 Interestingly
though, two independently isolated clones encoded Rtg3, a well
characterized transcriptional activator protein. Rtg3-68 (residues 305 to 486) and Rtg3-36 (residues 326 to 486) interact equally well with
LexA-Cyc8 in a two-hybrid assay (not shown), and both contain sequences
from the C-terminal region of Rtg3, known to be the activation domain
of the protein (28, Fig. 2).
To further investigate the Cyc8-Rtg3 interaction, we used a GST
affinity chromatography assay, in which 35S-labeled
Rtg3-68 protein was synthesized in vitro and was incubated with agarose bead-immobilized GST or GST-Cyc8 hybrid protein expressed in and purified from bacteria. As shown in Fig.
3, Rtg3 is specifically retained on the
GST-Cyc8 column (lane 3) but not on the column containing
GST alone (lane 2), strongly suggesting that Rtg3 directly associates with Cyc8 in the absence of any other yeast protein.
Rtg3 activates the transcription of CIT2, a gene encoding a
peroxisomal isoform of citrate synthase, and probably the transcription of additional genes involved in peroxisome biogenesis (29). Thus, we
subsequently explored the function of Cyc8 and Tup1 in the context of
the natural CIT2 promoter.
Dual Function of Cyc8-Tup1 on CIT2
Transcription-CIT2--
Transcription is induced by mitochondrial
dysfunction, and this regulatory pathway, through which nuclear gene
transcription responds to the functional state of mitochondria, is
termed retrograde regulation (24, 30). Both basal expression and
retrograde response of CIT2 is mediated by the Rtg3
transactivator, which is always bound to the CIT2 UAS
(29).
A typical retrograde response of CIT2 gene transcription is
shown in Fig. 4A.
CIT2 expression is much higher in wild type cells growing
under conditions of mitochondrial dysfunction (lane 2),
compared with the basal level of expression observed in normally growing cells (lane 1). However, basal expression and
retrograde response of CIT2 transcription are dramatically
reduced in a strain carrying a chromosomal deletion of CYC8
(lanes 3 and 4), suggesting that CIT2
is positively regulated by Cyc8. On the other hand, basal expression of
CIT2 is increased in a tup1 Distinct TPR Motifs of Cyc8 Interact with Tup1 and
Rtg3--
Two-hybrid assays performed in cyc8
To test whether the TPR domain is sufficient to recruit Cyc8-Tup1 to
the CIT2 promoter we performed a band shift experiment using
whole yeast protein extracts and a probe encompassing two Rtg3/Rtg1-binding sites (Rtg3 binds DNA as a heterodimer with Rtg1,
another bHLH/Zip protein, see "Discussion"). In agreement with
previous data (29), a stable low mobility complex was detected in the
presence of protein extracts derived from a wild type strain (Fig.
6, lane 2). The formation of
this complex is dependent on the presence of Cyc8, because extracts
from a cyc8 The Cyc8 C-terminal Domain Is Essential for Stimulation of CIT2
Transcription--
The TPR domain of Cyc8 provides sufficient Cyc8
function for transcriptional repression by bringing Tup1 to specific
DNA-binding proteins, while the C-terminal domain is dispensable.
Because we showed that Cyc8-Tup1 activates CIT2, we examined
whether recruitment of the complex by the TPR domain is sufficient for
positive regulation of CIT2 transcription. For this purpose,
derivatives of Cyc8 capable of interacting with both Rtg3 and Tup1,
either containing or lacking C-terminal sequences, were expressed in a
cyc8 In this study we provide direct evidence for a dual role of
Cyc8-Tup1 in transcriptional control. We found that besides the well
established repression activity, which is performed by Tup1, the
Cyc8-Tup1 protein complex can also act as a transcriptional co-activator, and this function is predominantly mediated by the Cyc8
protein. When the Tup1 repression activity is impaired, as it is in a
sin4 or a rgr1 mutant strain, Cyc8-Tup1 activates
an artificial reporter gene, and in response to specific metabolic signals, activates the transcription of the natural CIT2 gene.
Transcription of CIT2 is controlled by Rtg3 and Rtg1, both
members of the bHLH/Zip family of DNA-binding proteins. Recombinant Rtg3 and Rtg1 bind as a heterodimer at two sites within an upstream activation sequence of the CIT2 gene termed UASr
(31, 32). Heterologous promoters bearing a UASr respond to
mitochondrial dysfunction in a Rtg1/Rtg3-dependent manner
indicating that UASr is sufficient to mediate
CIT2 regulation (30). Notably, EMSAs using whole yeast
extracts (instead of recombinant Rtg1 and Rtg3 proteins) suggested that
additional yeast proteins, probably co-activators or co-repressors,
associate with the DNA-bound Rtg1-Rtg3 heterodimer (29). Our results
show that Cyc8 and Tup1 are indeed components of this
UASr-bound protein complex. The Cyc8-Tup1 complex
associates with Rtg3 in vivo and in vitro, and a
DNA fragment encompassing two Rtg3/Rtg1-binding sites
(UASr) gives rise to a stable protein-DNA complex, the
formation of which is dependent on the presence of the Cyc8 TPR domain.
Consistently, Rtg3 interaction is mediated by two specific TPR regions
of Cyc8, TPR4 to TPR7 and TPR8 to TPR10. A third separate region, TPR1
to TPR3, associates with Tup1 (18, 33) explaining how tandem TPRs can
link different proteins and assemble multiprotein complexes. Based on
these observations, we propose that recruitment of Cyc8-Tup1 to the
CIT2 promoter is mediated by specific TPRs that are
responsible for linkage of Cyc8 to Tup1 and of that complex to the
Rtg1/Rtg3 heterodimer.
Several lines of evidence suggest that Cyc8-Tup1 is directly involved
in the activation of CIT2 transcription and that this function is performed by the Cyc8 subunit. First, Cyc8-Tup1
specifically associates with the activation domain of Rtg3. This region
of Rtg3 has been shown to be the major activation domain of the
Rtg1/Rtg3 heterodimer because Rtg1, which does not possess independent
transactivation properties, functions to recruit Rtg3 to its binding
site (28). Thus, a possible role of the Rtg1/Rtg3 activation domain is
to simply contact the Cyc8-Tup1 complex. Second, deletion of
CYC8 or deletion of both CYC8 and TUP1
(data not shown) severely reduces the levels of CIT2
mRNA under inducing conditions of mitochondrial dysfunction. Under
these conditions, CIT2 transcription is defective even in
the presence of Cyc8 derivatives (N300 and N597) that fully complement
all known cyc8 Our data indicate that CIT2 transcription requires the
C-terminal domain of Cyc8, and in fact, this is the only case that a
function has been assigned to this region. When bound upstream of a
test promoter through a heterologous DNA-binding domain this C-terminal
region of Cyc8 does not activate transcription (data not shown);
therefore it does not function as a typical activation domain, but
rather plays a regulatory role. Cyc8 is a phosphoprotein, and specific
regions within this C-terminal domain, rich in serine and threonine
residues, are potential phosphorylation sites (16). Similarly, Rtg3
contains a serine/threonine-rich region which might also play a
regulatory role (28). Thus, it is conceivable that specific
modifications of these protein domains, such as phosphorylation or
dephosphorylation, may in fact modulate the transcriptional activity of
Cyc8-Tup1. Some of these modifications, particularly in the C-terminal
domain of Cyc8, are likely to be specific for the retrograde response
of CIT2 as this domain is dispensable for the regulation of
all other Cyc8-Tup1 repressible genes.
The signal(s) that mediate induction of peroxisomal genes upon
mitochondrial dysfunction are presently unknown, and although several
possible models can be envisaged using the available data, the
molecular mechanism by which Cyc8-Tup1 is converted from a co-repressor
to a co-activator of CIT2 is not yet understood. One model
predicts that, upon induction, Tup1 dissociates from the complex thus
unmasking Cyc8 activation potential. However, EMSAs performed with
protein extracts derived from either normal or mitochondria defective
cells detect neither quantitative nor qualitative differences on
UASr DNA-protein complexes (21,
29).4 This observation also
argues against the model according to which additional positive
regulatory factors associate with Rtg3/1-bound Cyc8-Tup1 assembling an
activator complex, although transient interactions with such factors
cannot be excluded. Another, more plausible, model postulates that in
response to specific signaling, Cyc8-Tup1 undergoes post-translational
modifications which could reveal the intrinsic activation potential of
Cyc8. Masking of Tup1 repression, although possible, cannot solely
account for the activation function of the complex because
Cyc8-mediated CIT2 induction is observed even in the absence
of Tup1 (tup1, a sin4
, or a rgr1
strain, suggesting that transcriptional activation is an intrinsic
property of the Cyc8-Tup1 co-repressor. In support of this notion we
demonstrate that Cyc8-Tup1 has a dual function on CIT2, a
gene encoding a citrate synthase that is expressed upon mitochondrial
dysfunction. First, we show that Cyc8-Tup1 is tethered to
CIT2 promoter by interacting with the activation domain of
Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2. Next
we demonstrate that Cyc8-Tup1 activates CIT2 transcription
in response to mitochondrial dysfunction, and this stimulatory effect
is mediated by Cyc8. In contrast, basal (noninduced) expression of this
gene is inhibited by Tup1. These findings establish a positive role for
the Cyc8-Tup1 complex in transcription and support a model by which
specific metabolic signals may convert the Cyc8-Tup1 transcriptional
co-repressor to a co-activator of certain promoters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
gene expression in response
to virus induction is also responsible for post-induction turn off
(35).
or a
rgr1
mutant strain. Taken together, these data suggest an
inherent potential of the Cyc8-Tup1 co-repressor for transcriptional
activation function and establish a dual role (positive and negative)
of this complex in transcriptional control.
EXPERIMENTAL PROCEDURES
ura3-52 trp1-
63
leu2::pet56 his3-
200). The two-hybrid screening was
performed in the strain L9FT5, which was constructed by replacing
his3-
200 with the L9His3 allele.
L9His3 promoter contains a single synthetic and perfectly
symmetric LexA-binding site in place of the Gcn4 UAS (8).
cyc8
and tup1
alleles have been described
previously (8). The sin4
allele was constructed by
inserting HIS3 between the internal PvuII and
PstI sites, while the rgr1
allele was constructed by inserting HIS3 between the NdeI
and NsiI restriction sites. sin4
and
rgr1
strains were generated by one-step gene replacement
using linear DNA fragments and were confirmed by Southern analysis.
-galactosidase activity after
retransformation into L9FT5 strain. Sequencing analysis revealed seven
different ORFs encoding proteins capable of two-hybrid interaction with Cyc8.
strains were isolated as described previously (24),
and protein concentrations were determined by the Bradford assay. A
CIT2 promoter region (
530 to +1) was amplified from L9FT5
genomic DNA by polymerase chain reaction, and a
BssHII-AatII fragment of it (106 base pairs) containing the two Rtg1/Rtg3-binding sites, was end-labeled by standard
methods using "Klenow" DNA polymerase and purified by Sephadex G-50
chromatography (Amersham Pharmacia Biotech). EMSA reactions were
performed with 10 µg of protein extract, 10,000 cpm of
CIT2 probe, 4 µg of poly(dI/dC) competitor DNA, 1 mM dithiothreitol, 10 mM Tris-HCl, 100 mM KCl, 2 mM MgCl2, and 5%
glycerol in a final volume of 20 µl. They were incubated at 4 °C
for 25 min, and samples were separated on a 5% polyacrylamide, 1× TBE
gel at 4 °C, 250 V for 2.5 h and visualized by autoradiography.
-Galactosidase assays were performed on
yeast cultures grown in the appropriate media and harvested during
early log phase (A600 < 1.0). Cells were washed
with 20 mM Tris (pH 7.5), 1.0 mM EDTA in order
to disperse the clumpy cyc8 and tup1 cells. LacZ values normalized to a600 represent the average of at least
three independent transformants, and they are accurate to 20-30%.
RESULTS
1, sin4
, and rgr1
strains
were co-transformed with plasmids carrying genes that express LexA-Cyc8
and the GAL1-LacZ reporter, and transformants
were assayed for
-galactosidase activity. As shown in Fig.
1, LexA-Cyc8 represses transcription from
the reporter promoter when functional Tup1 is present (wild type
strain, line 1). However, in the absence of Tup1
(tup
1), LexA-Cyc8 stimulates transcription by 7-fold
(line 2). This result indicates that Tup1 not
only actively represses transcription (8) but it also antagonizes the
activation potential of Cyc8. This cannot be explained simply by an
intermolecular masking of Cyc8 by Tup1 because, similarly to the Cyc8
protein alone, the Cyc8-Tup1 protein complex can also activate
transcription when the repression activity of Tup1 is impaired. In
sin4
or rgr1
strains, in which the
respective components of the holoenzyme mediator complex that are
essential for the establishment of the Tup1 repression activity are
missing, the LexA-Cyc8/Tup1 protein complex is converted to a
transcriptional activator (line 3 and
line 4). Noticeably, activation by LexA-Cyc8/Tup1 in these mutant strains is virtually higher (9-fold) than that in the
tup1
strain. These observations suggest that Cyc8 has the
potential to act as a transcriptional co-activator and that this
function is performed more efficiently in the context of the Cyc8-Tup1
protein complex.
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Fig. 1.
Transcriptional activation by LexA-Cyc8.
-Galactosidase activities (average of three independent
transformants) of wild type (WT), sin4,
rgr1, and tup1 deletion strains expressing
LexA-Cyc8. The LacZ reporter plasmid contains a minimal
promoter with LexA-binding sites upstream of a TATA box. For each case
a schematic representation of the proteins involved is indicated.
C, Cyc8; T, Tup1; S, Sin4;
R, Rgr1; polII Holo, RNA polymerase II
holoenzyme.
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Fig. 2.
Structure of Rtg3 and derivatives. The
structure of the Rtg3 protein (486 amino acids) is schematically
represented, including the bHLH/Zip motif (residues 284 to 374) and the
serine/threonine-rich region (S/T, residues
176-282). The C-terminal activation domain comprises the sequence 375 to 486. Both Rtg3-68 (residues 305-486) and Rtg3-36 (residues
326-486) contain the C-terminal activation domain of the protein and
lack most of the DNA-binding domain.
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Fig. 3.
Cyc8-Rtg3 interaction in
vitro. 35S-Labeled Rtg3 (residues 326-486)
interacting with a GST-Cyc8 hybrid protein or GST alone immobilized in
Sepharose beads. Lane labeled input contains only 20% of
the amount of the protein that was incubated with the beads.
strain (lane 5) indicating that CIT2 transcription is yet another
target of the Tup1 repression activity. In the tup1
strain, retrograde response appears to be comparable, although slightly
lower, than in wild type strain (lane 6). Thus, Tup1
inhibits basal expression of CIT2 but it might also be
required along with Cyc8 for maximal CIT2 induction. In
agreement with this notion, CIT2 expression is fully
de-repressed in Tup1 repression defective strains, such as
sin4
and rgr1
(lanes 7 and
8), in which Tup1 is expressed normally. In fact, the
expression levels of CIT2 in these mutant strains are
comparable with the levels observed under conditions of mitochondrial
dysfunction. Taken together, these results strongly suggest that
Cyc8-Tup1 has a dual function on the CIT2 promoter; it
inhibits basal transcription, but moreover it acts as a co-activator that mediates retrograde response. It is noteworthy that Rtg3, which is
the limiting factor for CIT2 transcription (28), is present
at equal levels in wild type, cyc8
, and
tup1
cells growing either at normal or at inducing
conditions (28, 29, and data not shown).
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Fig. 4.
Normal expression of the CIT2
gene requires functional Cyc8 and Tup1. A, total
RNA was extracted from cultures of wild type (WT),
cyc8, tup1, sin4, and rgr1
deletion strains exponentially grown in the indicated conditions
(N, normal; MD, mitochondrial dysfunction). RNA
was fractionated in formaldehyde agarose gel (1.5%), transferred to
nylon membrane, and hybridized with probes specific for CIT2
and TBP (used to normalize RNA levels) as described under
"Experimental Procedures." B, Cyc8 deletion derivatives
(shown in Fig. 5), were expressed in a cyc8 deletion strain,
and yeast transformants were cultured in normal (N) and
mitochondrial dysfunction (MD) conditions. Total RNA was
extracted, fractionated in a formaldehyde-agarose gel, transferred to
nylon membrane, and probed with CIT2 and TPB specific
probes.
and
tup1
strains indicated that Rtg3 specifically interacts
with Cyc8 even in the absence of Tup1, while Tup1 interacts with Rtg3
only in the presence of Cyc8 (data not shown). The TPR domain of Cyc8
mediates protein-protein interactions and was proposed to link specific
DNA-binding proteins to Tup1 (18). Thus, we examined whether Rtg3
interacts with specific TPR motifs of Cyc8 by testing various deletion
derivatives of Cyc8 for Rtg3 interaction in a two-hybrid assay (Table
I and Fig.
5). N175, that contains only three
N-terminal TPRs (TPR1 to TPR3), does not activate transcription of the
LacZ reporter demonstrating its failure to interact with
Rtg3. In contrast, N300 that contains TPR1 to TPR7 strongly interacts
with Rtg3 (it activates transcription over 90-fold), indicating that
interaction with Rtg3 is mediated by specific TPR motifs, probably TPR4
to TPR7. However, the internally deleted Cyc8 derivative
175-281 that lacks TPR4 to TPR7 but maintains TPR8 to TPR10 also interacts with
Rtg3 as judged by its activity on the LacZ reporter, which is stimulated over 30-fold. Finally, derivatives such as C560, which
comprise the C-terminal domain of Cyc8 but lack TPR sequences, are
completely inactive. These data suggest that Rtg3 interacts with at
least two independent combinations of TPR motifs, TPR4-TPR7 and
TPR8-TPR10. It should be noted that none of these regions overlap with
the Tup1 interaction domain, which consists of TPR1 to TPR3 (Ref. 18
and Fig. 5), and it explains how Rtg3 and Tup1 (which do not interact
directly) can simultaneously associate with the TPR domain of Cyc8.
Two-hybrid assays for Rtg3-Cyc8 interaction
-Galactosidase activities (average values from three independent
transformants) in cells expressing the indicated hybrid proteins. The
LacZ reporter plasmid contains four overlapping LexA binding sites
upstream of the GAL1 TATA box. Values are accurate to
±30%. Fold activation represents the ratio of
-galactosidase
activities in strains containing Rtg3 fused to the activation domain of
Gal4 (Rtg3-Gal4) versus those containing Gal4 only.
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Fig. 5.
Structure and function of Cyc8 deletion
derivatives. The structure of Cyc8 (966 amino acids) along with
deletion derivatives are schematically shown. Numbers
represent TPR motifs. For each derivative the following phenotypic
properties are indicated: Rtg3 interaction (Table I), Tup1 interaction
(18), and CIT2 retrograde response (Fig. 4B).
Phenotypes are defined as follows: +, wild type; ±, partial function;
, functionally indistinguishable from cyc8 allele.
strain do not give rise to shifted bands
(lane 3). Moreover, ectopic expression of Cyc8 in the
cyc8
strain restores complex formation (lane
4), and more importantly, the complex is formed even by expressing only the TPR domain of Cyc8 (lane 5). These results strongly
suggest that protein-protein interactions mediated by TPR motifs are
sufficient to recruit Cyc8-Tup1 to the CIT2 promoter.
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Fig. 6.
The TPR domain is sufficient for recruitment
of Cyc8-Tup1 to the CIT2 promoter. Total yeast protein was
extracted from wild-type and cyc8 strains or from a
cyc8
strain expressing either full-length Cyc8 protein or
the N300 derivative that contains only TPRs (see Fig. 5). EMSAs were
performed using a DNA fragment that contains the two Rtg1/Rtg3-binding
sites of the CIT2 promoter.
strain, and CIT2 mRNA levels were
analyzed by RNA blotting. As shown in Fig. 4B and summarized
in Fig. 5,
175-281, which contains the entire C-terminal domain of
the protein, supports wild type levels of CIT2 transcription
(lanes 7 and 8). In contrast, N300 and N597 that
lack the C-terminal domain are inactive (lanes 3-6),
despite their ability to interact with Rtg3 and to complement all
previously described cyc8
defects (18). Finally, a longer
derivative, N816, that contains most of the C-terminal domain is only
partially functional (lanes 1 and 2). These
results indicate that Rtg3 interaction alone is not sufficient for
transcriptional activation of CIT2; normal retrograde
response of CIT2 expression requires the C-terminal domain
of Cyc8 as well. To our knowledge, retrograde regulation is the only
case where a specific function has been assigned to this domain of
Cyc8, most likely reflecting the unique regulatory mode of Cyc8-Tup1
action on the CIT2 promoter.
DISCUSSION
-specific phenotypes, including slow growth
and temperature-sensitive lethality (18). These results suggest that
lower CIT2 transcription is not an indirect physiological
effect of the cyc8
mutation. In agreement to this, deletion of TUP1, which causes similar pleiotropic defects
as cyc8
, does not significantly affect the levels of
CIT2 mRNA under inducing conditions. Third, when brought
to a LexA operator-containing reporter, LexA-Cyc8 activates
transcription in a tup1
strain. Similarly, the
LexA-Cyc8/Tup1 protein complex activates transcription in an isogenic
sin4
or a rgr1
strain that lacks the
respective factor essential for Tup1 repression. These data, together
with previous observations (26, 27), establish a positive role of Cyc8
in transcription and moreover they suggest a dual, positive and
negative, function of the Cyc8-Tup1 protein complex on CIT2. Indeed Cyc8-Tup1 inhibits the basal (uninduced) expression of CIT2, and this function is performed by Tup1.
CIT2 transcription is derepressed in cells carrying the
tup1
mutation while in cells that express Tup1, but lack
Sin4 or Rgr1, CIT2 derepression occurs at even higher
levels. This observation further suggests that Cyc8 activation function
is better performed in the context of the Cyc8-Tup1 protein complex.
strain, Fig. 4A). Regulatory
mechanisms by which proteins undergo conformational changes and
activate transcription have been previously reported, and in some
cases, as that of the retinoic acid receptor, these mechanisms have
been characterized extensively (34). It is conceivable that Cyc8
undergoes specific structural changes, i.e. by
phosphorylation of the C-terminal domain, which is specifically
required for stimulation of CIT2 transcription, and this
could possibly be the key step through which the complex attains its
activation potential. It must be emphasized that according to this
model post-translational modifications of Cyc8 and probably of Tup1
have such an effect only when the complex is associated with the Rtg3/1
proteins. This hypothesis explains why Cyc8-Tup1 has a dual function
only in CIT2 expression and why DNA-bound LexA-Cyc8/Tup1 is
not converted to an activator of a synthetic reporter under inducing
conditions of mitochondrial dysfunction (data not shown). Finally, it
is known that Cyc8-Tup1 functionally interacts with the basic
transcription machinery as well as with chromatin (15); thus it is
conceivable that Cyc8 might activate CIT2 transcription by
affecting either one or even both processes.
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ACKNOWLEDGEMENTS |
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We thank George Thireos, Maria Monastirioti, Despina Alexandraki, and Tassos Economou for helpful discussions, D. Alexandraki for providing the two-hybrid library DNA, and Eleftheria Vrontou for RNA blotting experiments.
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FOOTNOTES |
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* This work was supported by the Greek Ministry of Research and Technology (PENED) and by a Training and Mobility of Researchers grant from the European Union (to D. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-391162; Fax:
81-391101; E-mail: Tzamarias{at}imbb.forth.gr.
2 A. Ramne and P. S. Sunnerhagen, unpublished data.
3 D. Tzamarias, unpublished observations.
4 R. S. Conlan and D. Tzamarias, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TPR, tetratrico peptide repeat; UAS, X-Gal, 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; UAS, upstream activation sequence..
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REFERENCES |
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