![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Natural Sciences Section, Södertörns Högskola, and Department of Biosciences, Karolinska Institute, Huddinge, Sweden
Received 16 September 2004/ Accepted 11 October 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Tup1-Ssn6
complex is recruited to the promoters of target genes by interaction
with DNA bound transcriptional repressor proteins that recognize
specific sequences within target gene promoters. Examples of such
repressor proteins include the Mat
2 repressor that regulates
mating-type-specific genes
(19) and the Mig1
repressor that regulates glucose-repressed genes
(35), as well as Crt1 and
Sko1, which are involved in the control of DNA repair
(15) and hyperosmotic
stress (28),
respectively. Recently, this gene-specific role has been complemented
by observations that Tup1 might be involved in establishing domains of
heterochromatin structure in the subtelomeric regions of chromosomes
(31). These so-called
HAST domains contain clusters of Tup1 and Ssn6 repressed genes and
coincide with regions that are deacetylated by the histone deacetylase
Hda1. It has thus been suggested that Tup1-Ssn6 establishes formation
of heterochromatin in these regions by recruiting Hda1. HAST domains
are distinct from adjacent heterochromatin regions that are established
via Tup1-independent recruitment of the Sir2 histone
deacetylase.
In the evolutionarily distant fission yeast
(Schizosaccharomyces pombe) there are a number of differences
in the mechanisms involved in gene-specific repression and the
formation of transcriptionally silent regions of heterochromatin
compared to S. cerevisiae. Most notably, fission yeast have an
interfering RNA (RNAi)-based mechanism for establishment
and maintenance of silent heterochromatin similar to other eukaryotes
that is apparently absent from S. cerevisiae
(11,
40). In spite of these
differences, the Tup1-Ssn6 corepressor is conserved throughout fungi
and there are also related corepressors in higher eukaryotes such as
HIRA, TLE1, and Groucho, which are functionally homologous to the yeast
corepressor (6,
7). The fission yeast is
unusual compared to other fungi because it contains two genes encoding
Tup1-like corepressors. Evidence that the S. pombe proteins
function similarly to the Tup1-Ssn6 complex in S. cerevisiae
has been reported recently
(12,
14,
16,
23,
24,
27,
45). Tup11 has been shown
to repress target genes containing LexA binding sites when fused to the
LexA DNA-binding domain, and binding studies have shown that Tup11 is
able to interact with the S. cerevisiae transcription factor
Mat2 (24).
Studies with Tup11 and Tup12 have also revealed that they both act as
negative regulators of the S. pombe
fbp1+ gene in a redundant fashion
(16). As in S.
cerevisiae, the tup11– and
tup12– deletion phenotypes are
also associated with flocculation in liquid media, defective mating,
and defects in stress responses. Examples of known target genes for
Tup11 and Tup12 are the intracellular cation transporter
cta3+, which is activated under
high-salt conditions
(12). Tup11 has also been
reported to interact directly with Fep1, a transcription factor that
represses the expression of the iron transport genes
fio1+ and
fip1+ in response to high iron
concentrations (27,
45).
Gene duplication is thought to be an important mechanism involved in the evolution of biological diversity. The apparent gene duplication that has given rise to the tup11+ and tup12+ genes, located close to the end of chromosome 1 in fission yeast, therefore offers an interesting opportunity to study this process. As expected, Tup11 and Tup12 have been shown to play at least partly redundant roles. However, no comparative study has been made to determine whether and to what extent the significant sequence divergence between the two proteins is associated with differences in their function. We show here that both proteins have retained the ability to interact with each other and with the fission yeast Ssn6 protein but that Tup12 has evolved a specialized role in the regulation of the stress response.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Western blotting and coimmunoprecipitation.
Chromosomally tagged yeast strains
were grown in YES medium for 18 h to mid-log phase
(107 cells/ml). Cells were harvested and washed in NP-40
lysis buffer (0.150 M NaCl, 0.050 M Tris [pH 8], 1%
NP-40 with protease inhibitors [Complete cocktail inhibitors;
Roche catalog no. 1873580], leupeptin at 2 µg/ml, and 1 mM
phenylmethylsulfonyl fluoride). Cell extract was made with glass beads
in a bead beater. For immunoprecipitation experiments, the extract was
precleared with protein A (Sigma catalog no. P-1406) coupled beads and
incubated with a polyclonal rabbit -GFP antibody (Clontech
catalog no. 8372-2). For 500 µl of extract, 1 µl of
-GFP antibody was used. The antibodies were precipitated with
protein A-coupled beads as described above. The precipitated beads were
washed three times and resuspended in sodium dodecyl sulfate (SDS)
loading buffer. Resuspended protein extracts were resolved on
SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western
blot analysis. The membrane was probed with monoclonal mouse
-HA antibody (Roche catalog no.
12CA5).
Immunofluorescence microscopy.
Chromosomally
double-tagged strains were grown for 18 h to mid-log phase
(107 cells/ml), harvested, fixed with formaldehyde, and
incubated with primary and secondary antibodies as described previously
(3). The following primary
antibodies were used: -HA monoclonal mouse (Roche catalog no.
12CA5) and -GFP polyclonal rabbit (Molecular Probes catalog
no. 11121). Secondary antibodies conjugated with Texas red or
fluorescein isothiocyanate (Jackson Immunoresearch Laboratories) were
used. Nuclear staining was performed with DAPI
(4',6'-diamidino-2-phenylindole). The cells were
subjected to confocal microscopy with a Zeiss Axioscope II microscope.
Digital deconvolution was performed with Openlab software by using
0.3-µm z spacing and nearest-neighbor
deconvolution.
Protein alignments and phylogenetic clustering. Protein sequences were subjected to CLUSTAL W alignment with Macvector 6.5 software by using the CLUSTAL W blosum matrix with a gap penalty of 20. The following sequences were handled and analyzed: S. cerevisiae, Tup1 (P16649); Saccharomyces paradoxus, Tup1 (MIT_Spar_c90_2709); Saccharomyces mikate, Tup1(Smik_Contig2778.4); Saccharomyces bayanus, Tup1 (Sbay_Contig666.45); Saccharomyces kudriavzevii, Tup1 (Skud_Contig1764.4); Saccharomyces castellii, Tup1 (ScTup1 as_Contig629.12); Kluyveromyces lactis, Tup1 (P56094); Candida albicans, Tup1 (P56093); Magnaporthe grisea, Tup1 (AACUO1001716.1); Neurospora crassa, Tup1 (P78706); Emericella nidulans, Tup1 (AAB63194; Yarrowia lipolytica, Tup1 (CAC81004; Dictostelium discodeum, Tup1 (AAC29438; S. pombe, Tup11 (Q09715); and S. pombe, Tup12 (Q9UUG8). Full-length protein sequences were used in Fig. 1A and B. The cross comparison in Fig. 1C was generated by comparing the different Tup1 domain as defined in Fig. 1B.
|
tup11 and tup12 strains were grown
in YES medium at 30°C for 18 h to early mid-log phase
(0.5 x 107 cells/ml), followed by a medium change
for 45 min. For normal conditions the cells were
transferred to YES medium, and for stress induction the cells were
transferred to YES medium containing 1 M KCl for 45 min. Cells were
harvested and immediately placed in lysis buffer and phenol. Two RNA
samples from independent cultures were prepared for each condition, and
25 µg of RNA was subjected to reverse transcription (RT;
11904-018 Invitrogen) and labeled with CY3 (CY3 dCTP 53021; Amersham)
or CY5 (CY5 dCTP 55021; Amersham) prior to hybridization on S.
pombe gene microarrays from Eurogentec SA, Seraing,Belgium (42). Altogether
four microarrays spotted in duplicate were used, which generated four
datum points for each condition and gene. The microarray signals were
visualized with a ScanExpress laser scanner and quantified with the
Imagene 4.2 software. The data were analyzed and normalized with the
Lowess per-spot per-chip method with the GeneSpring software (Silicon
Genetics). Data with a standard deviation of >80% of the
mean value were excluded. To assess the significance of the
twofold differences identified, we performed a one-sample, one-tailed
t test to test the null hypothesis that observed changes are
not significant. Stress data for the individual Tup11/12 differentiated
genes and control groups were extracted from the Sanger Institute
database
(www.sanger.ac.uk/PostGenomics/S_pombe/projects/stress/)
(5). To calculate the
enrichment of genes found in core environmental stress response
(CESR) clusters and to determine the significance for
observed results by chance, we used the hypergeometric distribution
test in the GeneSpring
software.
RT-PCR.
RNA from JY741,
JY741(tup11), JY741(tup12),
Y741(tup11,12) was extracted
independently as described above and was subjected to DNase treatment
and RT (11904-018; Invitrogen) for synthesis of cDNA.
Samples were subjected to duplex PCR, and the generated products were
separated with electrophoresis on a 1.5% agarose gel and
visualized with ethidium bromide. Gels were digitalized and quantified
with the Imagauge 4.0
software.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The fungal Tup proteins are similar to each other throughout their protein sequence, and they share the same domain architecture (Fig. 1B). The proteins are all highly homologous in the WD40 C-terminal domain and less conserved in the N-terminal Ssn6-binding domain and the variable central domain, proposed to play a role in the repression activity of the protein. Sequence alignments of the individual domains show that these three different domains are differentially conserved in relation to evolutionary distance (Fig. 1C). For example, Tup11 and Tup12 that are most similar to each other overall show the lowest degree of similarity in the central domain of any of the species compared in Fig. 1C. This suggests that the different domains have evolved as separate functional units.
Relative expression levels, interaction, and localization of the Tup11 and Tup12 proteins. Since Tup11 and Tup12 are most conserved in the C-terminal domain that has been implicated in tetramerization of Tup1-like proteins, we investigated aspects significant to interaction between Tup11 and Tup12. Previous work (12) has shown that Tup11 and Tup12 can interact in coimmunoprecipitation experiments with extracts prepared from cells that overexpress the proteins. We created a strain in which the chromosomal loci encoding Tup11 and Tup12 were modified so that the proteins would be expressed in an epitope-tagged form from their endogenous promoters. Using cell extracts produced from this strain, we showed that at least a portion of Tup12-HA could be immunoprecipitated by using antibodies directed against Tup11-GFP (Fig. 2A). The ability of Tup11 and Tup12 to interact combined with the model in which Tup1-like proteins function in the form of a tetramer creates potential complications for the acquisition of independent functional roles by the two proteins during evolution. This is because if the two proteins are expressed at similar levels and freely associate, the vast majority of resulting tetramers would contain both Tup11 and Tup12. To measure the relative expression levels of the Tup11 and Tup12 proteins, we created modified strains in which the chromosomal loci have been modified to express Tup11 and Tup12, respectively, fused to the HA epitope tag. Extracts from these strains and a wild-type, untagged strain were analyzed by Western blotting. Figure 2B shows that there is no large difference in the expression level of the Tup11 and Tup12 proteins. We reasoned that if the vast majority of Tup1-like oligomers contain both Tup11 and Tup12, there would be a high degree of colocalization of the two proteins in the nuclei of cells. To test this, we studied the strain in which both tup11+ and tup12+ loci were tagged with the HA and GFP epitope tags, respectively, by using immunofluorescence microscopy (Fig. 2C). Images from immunostained cells were captured and deconvolved. Both proteins are distributed in a somewhat punctate fashion throughout the nucleus, and there is a significant degree of overlap in their distribution. However, it was clear that the two proteins were not completely colocalized since there were nuclear regions in which each protein was found in the absence of the other. We conclude that there are regions within the nucleus that exclusively contain either Tup11 or Tup12 but not both.
|
|
, tup12, and
double-knockout tup11,12 deletion phenotypes
under various conditions that have been reported to affect the growth
of tup11,12 deletion strains
(12,
16).Interestingly, we observed differential phenotypes for the
tup11 and tup12 deletion strains on medium containing
1 to 1.5 M KCl and 0.25 M CaCl2 (Fig.
4A and
B). Figure 4A shows that cells
lacking Tup12 grow equally poorly on KCl as cells lacking both Tup
proteins. Thus, under these conditions there are at least some Tup12
functions that are important for growth that cannot be performed by
Tup11. The same is true for growth on 0.25 M CaCl2 (Fig.
4B). However, at lower
levels of CaCl2 (0.1 M), Tup11 can replace the critical
functions of Tup12, and only the double mutant shows a reduced growth
phenotype. These observations support the view that Tup11 and Tup12 are
functionally redundant under some conditions but that under other
conditions they are functionally diverged. Since there was a
theoretical possibility that the phenotypes we observed for
tup12 mutant could be due to secondary modifier mutations in
the strain that we used, we backcrossed the mutant to a wild strain to
test for cosegregation of the salt sensitivity phenotype with the
mutated tup12 locus. Cosegregation was observed in all spores
analyzed from the cross (data not shown). To further investigate this
issue, we overexpressed Tup11 and Tup12 in a tup12
background to see whether expression of either protein could rescue the
phenotype associated with the tup12 deletion. Expression of
Tup12 from a plasmid rescued the KCl and CaCl2 sensitivity
phenotypes associated with defects in Tup12, as expected (Fig.
4C). However,
overexpression of the wild-type tup11+
allele from the same plasmid could not complement the phenotype of
tup12 strains. We therefore conclude that at least
some critical aspects of CaCl2 and KCl adaptation are
associated with a specific function of the Tup12
protein.
|
tup11 and tup12 mutant strains
under both normal (YES at 30°C) and KCl-stressed (YES at
30°C incubated with 1 M KCl for 45 min) growth conditions. RNA
samples were prepared from each culture, and labeled probes were made
by incorporation of Cy3 and Cy5 fluorescent nucleotides into cDNA
during RT prior to hybridization to duplicate microarrays for each
growth condition. The duplicate pairs of RNA from
tup11 and tup12 strains were each
labeled in both alternative dye orientations to reduce putative dye
bias artifacts from the final combined set of data. The results of the
experiments in the absence or presence of KCl are summarized in the
ratio-intensity plots shown in Fig.
5. We detected 49 of 4,905 measurable genes under normal growth conditions
and 29 of 4,931 measurable genes under KCl stress that showed a
differential effect of >2-fold (red spots). Ten genes are
common between these two groups. A complete list of the differentially
regulated genes in Fig. 5
is available in the supplemental material. Tables
2 and 3 list twofold
differentially affected genes in the
absence and presence of KCl, respectively, for which the fold change values are
statistically significant (P < 0.05). Interestingly,
within these more stringently selected gene sets, there is a clear bias
toward genes whose expression is increased in the
tup12 strain relative to the tup11
mutant. This could be due to the fact that under the physiological
conditions studied here most of the reproducible differential effects
reflect genes that are specifically repressed by
Tup12.
|
|
|
tup12 strain, we selected a number of genes with this
behavior for validation both in the absence or presence of KCl-induced
stress. All nine of nine tested genes and conditions in which the
tup11/tup12 expression ratio was
<0.5 were validated (Tables
1 and
2). To determine whether
the ratios of <0.5 are due to a defect in repression by Tup12
or a hypothetical defect in activation by Tup11, it was necessary to
compare transcript levels for differentially altered genes in the two
mutant strains with their levels in wild-type and double-mutant
strains. In all cases tested, the expression difference was due to a
defect in Tup12-dependent repression (Fig.
6A). Figure 6B shows negative
controls confirming that the RT-PCR amplified bands reflect RNA levels
present in the samples. Generally, there was a good quantitative
correspondence with the ratio calculated from the arrays. We infer that
Tup12 plays a functionally diverged role in repressing a number of
genes under normal and KCl-induced stress conditions. The KCl
sensitivity associated with Tup12-defective strains could be due to
inappropriate expression of one or more of these genes during
KCl-induced stress.
|
strain. However, our results (to be published
elsewhere) show that ssn6+ is an
essential gene in fission yeast. Characterization of different tagged
strains that we have constructed showed that insertion of an HA tag at
the C terminus of ssn6+ leads to
stress phenotypes similar to those observed for Tup12-defective
strains. As shown in Fig.
6C, the ssn6-HA
strain grew less well than the wild type on 1 M KCl at 30°C,
and the growth defect was enhanced at 36°C. This phenotype is
specific to the ssn6-HA strain since it was not observed in
the ssn6-GFP strain at either temperature (Fig.
6C). Next, we wanted to
investigate transcript levels from Tup12-dependent genes in the
ssn6-HA strain by RT-PCR. Figure
6D shows that all of the
genes that were Tup12 dependent in Fig.
6A were also derepressed
in the ssn6-HA strain at 30°C. We conclude that the
Ssn6 protein is important for regulation of Tup12-dependent target
genes.
Genes differentially regulated by Tup11 and Tup12 are over-represented in genes induced by environmental stress.
We noted that several
of the genes that are differentially affected by
tup11 and tup12 are known stress
response genes. To investigate this further, we used the hypergeometric
distribution to test the significance of the overlap with the 237 CESR
genes identified previously
(5). The top 100
Tup12-specific genes identified in Fig.
5A shows a highly
significant (P = 10–16) overlap
with the CESR genes. A significant overlap(P = 0.03) was also obtained with the 24
genes in Table 2. The CESR
genes were defined as genes that responded >2-fold to at least
four of five stress conditions tested
(5). These were oxidative
stress (H2O2), heavy metal toxicity (Cd),
elevated temperature (39°C), osmotic stress (sorbitol), and DNA
damage (MMS). Figure
7A shows the frequency distribution for the number of stresses that induce
each of the Tup12-specific genes by >2-fold (note that 2 of 24
genes are omitted due to a lack of data). Only two genes were not
induced by any stress. The remaining genes appear to reflect two
distributions. The first consists of CESR genes, whereas the second is
made up of genes that respond more specifically to stress, most often
to only one stress condition. Figure
7B shows the frequency
distribution of stress responses for the non-CESR genes. Most frequent
are responses to H2O2, Cd, and heat, whereas
sorbitol-induced stress responses are less frequent. None of these
genes responded to MMS. We conclude that during the functional
divergence of Tup11 and Tup12 during evolution the two proteins adopted
overlapping but distinct roles and that Tup12 has become specialized in
the regulation of a subset of stress response genes. These genes
include both CESR genes and stress-specific response
genes.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The
redundant and distinct functions of Tup11 and Tup12 occur in highly
related physiological contexts. For example, our data show that on 0.1
M CaCl2 the two proteins appear to be functionally
redundant, whereas on 0.25 M CaCl2 Tup11 is completely
unable to rescue the nongrowth phenotype associated with defects
affecting Tup12. This could be accounted for either by a threshold in
the ability of Tup11 to correctly regulate a set of Tup12-regulated
genes as CaCl2 levels increase or by considering the two
conditions as physiologically separate with at least partially
different sets of Tup-targeted genes. The sensitivity of
tup12 mutants to KCl is more clear-cut. In this case,
we have not seen any rescue of the tup12 deletion phenotype by
Tup11 under any condition tested. These phenotypes are also manifested
by the ssn6-HA loss-of-function allele, suggesting that Tup12
functions together with Ssn6 in contexts in which it has a different
function from Tup11. The phenotypic differences caused by defects in
Tup11 and Tup12 in the present study are somewhat more pronounced than
those reported for similar conditions previously
(12). The differences may
reflect differences in the way the experiments were performed. It is,
however, important to note that the phenotypes we observed cosegregate
with the tup12 mutation through meiosis and that they
can be rescued by plasmid-mediated expression of Tup12 but
not by plasmid-mediated expression of Tup11.
The existence of diverged functions for Tup11 and Tup12 has to be viewed in the context of what is known about the structure of the Tup1-Ssn6 corepressor complex. A complicating feature here is that Tup1 in budding yeast is thought to build a tetrameric complex that binds to a single Ssn6 subunit. In fission yeast maximum diversification would be created if the Tup11 and Tup12 subunits were unable to participate in the same tetrameric structures and that these had a differential ability to interact with the fission homologue of Ssn6. Using coimmunoprecipitation of epitope-tagged proteins expressed at endogenous levels, we have confirmed the previous observation that Tup11 and Tup12 can interact together (12) even when they are expressed at normal levels. In addition, we have shown that there is no large difference in the affinity of either protein for Ssn6. Indeed, all of the Tup12-specific genes we tested also require Ssn6, strongly suggesting that Tup12-specific repression is mediated by Tup-Ssn6 corepressor complexes and not by a putative alternative mechanism in which Tup12 functions alone or together with an alternative partner protein. Thus, these assays do not detect a functional difference between the C-terminal oligomerization domain and the N-terminal Ssn6-interacting domains of these two proteins that are 41 and 76% diverged in sequence, respectively. Tup11 has in addition been shown to interact with Ssn6 in the two-hybrid system previously (24). Since Ssn6 is thought to be involved in targeting corepressor complexes to promoters (36, 37), our results suggest that the difference between Tup11 and Tup12 may be the result of differences in the repression activity of recruited complexes. Consistent with this the repression domains of the proteins are highly divergent in sequence with only 8% of residues conserved between them. Further studies will be required to address this issue. It is unclear whether the distinct repression activities of Tup11 and Tup12 are manifested by corepressor complexes containing a mixture of the two proteins or by complexes containing only Tup11 and Tup12. Since Tup11 and Tup12 are expressed at similar levels, one might predict that most corepressor complexes would contain a mixture of Tup11 and Tup12 subunits interacting with Ssn6. However, we cannot exclude the existence of components in vivo that favor the assembly of corepressors containing exclusively Tup11 or Tup12 proteins. Our immunofluorescence data suggest that such a mechanism may exist since the localization of the proteins in the nucleus is partially distinct (Fig. 2C).
Using DNA
microarrays we identified a number of genes that are differentially
affected by defects in Tup11 and Tup12. The most reproducible of these
showed a relative expression increase in tup12
compared to tup11 that could be validated with
RT-PCR. cta3+ and
hsp16+ are genes that are
repressed on normal medium by Tup12-Ssn6 but that are physiologically
derepressed by KCl-induced stress. On KCl, expression levels in the
mutant strains are similar to those seen in the wild type, and
therefore it is difficult to attribute the KCl sensitivity phenotype to
inappropriate regulation of genes with this behavior. SPCC1739.08c and
SPBC359.06 are normally repressed in the absence or presence of KCl,
and Tup12-Ssn6 defects cause derepression under both
conditions. Genes showing this pattern could cause
sensitivity to KCl if inappropriate expression of the genes on KCl
media has a more severe consequence for growth than in its absence.
sou1+ represents a third pattern
of regulation in which Tup12-Ssn6 specific repression is only seen in
the presence of KCl. Inappropriate expression of such genes could be
associated with the KCl sensitivity seen in the tup12
and ssn6-HA strains.
The RT-PCR experiments depicted in
Fig. 6 demonstrate that
pure Tup12-containing corepressors are fully active on Tup12-specific
genes because repression of all five genes tested is as efficient in
tup11 cells as in the wild type. However, the occurrence of
Tup11 in complexes regulating these genes in wild-type cells cannot be
excluded and, indeed, in the absence of Tup12, pure Tup11 complexes can
partially repress Tup12-specific genes. The partial repression effect
seen in tup12 cells could of course be due to a
Tup-independent repression function of the Ssn6 protein.
We cannot formally exclude that some of the genes shown in Fig. 5 are due to loss of an activator activity associated with Tup11. However, since all of the examples we have studied by RT-PCR are due to a loss of Tup12-specific repression activity, it is likely that the vast majority of these genes represent targets for Tup12-specific repression. This supposition is further supported by the fact that the differentially affected genes are enriched in genes that are known to be derepressed during environmental stress. Sequence analysis of the Tup12-dependent promoters also indicate the presence of ATF/CRE binding sites and T-rich elements at similar frequencies to those described earlier for the so-called CESR genes that are regulated >2-fold in at least four of the five stress conditions tested previously by others (5). We have not found any other DNA motifs that could account for Tup12-specific repression.
A small number of genes targeted by Tup11 and Tup12
have been characterized in fission yeast previously. The
fbp1+ gene has been shown to be a
target for both Tup11 and Tup12, which appear to function in a
redundant fashion to repress its expression
(16). Consistent with
this view, we did not identify
fbp1+ as a differentially affected
gene in our DNA microarray experiments. While scrutinizing a previous
report (27), we noticed
that the fio1+ gene, which encodes
one subunit of a bipartite iron transporter, is
partially derepressed in tup12 mutants but not in
tup11 mutants.
fio1+ was not detected with
statistical significance in our array data but showed a high
degree of Tup12 dependency in one experiment. The coregulated gene
encoding its dimerization partner,
fip1+, was however reproducibly
identified in our study. In our microarray and RT-PCR data, the
cta3+ gene encoding a cation
transporter is also heavily dependent on Tup12 for its repression.
However, derepression in a tup12- strain
was not seen in the previous study mentioned above
(12). The identification
of hsp16+ as a specific target of
Tup12-dependent repression is interesting in the light of previous
results. Tup12 has been shown to interact with Hsp16 in the two-hybrid
system (34), suggesting
the possibility that Hsp16 could participate in feedback regulation
of its own expression or in the expression of other
Tup12-repressed stress genes. Furthermore, together with
SPAC19C7.04c, hsp16+ has been
shown to be the target of RNAi-mediated repression
(29). Thus,
Tup12-specific attributes could represent evolutionary
adaptations that integrate Tup12-mediated repression with RNAi-mediated
silencing mechanisms. Recently, it has also been suggested that
ATF/CRE-regulated genes are involved in heterochromatization by an
alternative silencing mechanism involving components of the stress
signaling pathway (17,
18). It is possible that
Tup11/12-Ssn6 corepressors could be involved in such a
mechanism.
In budding yeast there are several different signal transduction pathways that mediate the response to different types of environmental stress. In fission yeast a large number of different stresses appear to elicit responses via a common signaling pathway that is dependent on the Sty1 protein. This has led to the question of how different stress conditions elicit different responses at the level of gene regulation (5). In the light of our results, it is possible that the different Tup proteins give specificity to gene targets associated with different environmental stresses. However, many of the genes we identified that differentially require Tup11 and Tup12 for their regulation are over-represented in genes that are regulated by most of the stresses for which microarray data are available (H2O2, Cd, heat, sorbitol, and MMS) (5). Thus, the Tup12-specific mechanism is probably a component of the general stress response. Indeed, the set of genes we identify here also shows an overlap with the CESR genes. Importantly though, the majority of the Tup12-specific genes are induced by a more restricted range of stress conditions and are not CESR genes. In such cases, Tup12 and Ssn6 might contribute to specificity of the stress response. One example is cta3+ where Tup corepressors have recently been reported to play a role in stress response specificity by creating an appropriate chromatin structure in the promoter region (13). It would be interesting to determine whether these effects are mediated specifically by Tup12.
|
ACKNOWLEDGMENTS |
|---|
This research was funded by project grants from the Swedish Research Council and the Foundation for Strategic Research. A.P.H.W. is a senior investigator financed by the Swedish Research Council.
|
FOOTNOTES |
|---|
Supplemental
material for this article may be found at
http://mcb.asm.org/.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bahler, J., J. Q. Wu, M. S. Longtine, N. G. Shah, A. McKenzie III, A. B. Steever, A. Wach, P. Philippsen, and J. R. Pringle. 1998. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943-951.[CrossRef][Medline]
3. Bjerling,
P., R. A. Silverstein, G. Thon, A. Caudy, S. Grewal, and K.
Ekwall. 2002. Functional divergence between histone
deacetylases in fission yeast by distinct cellular localization and in
vivo specificity. Mol. Cell. Biol.
22:2170-2181.
4. Carrico,
P. M., and R. S. Zitomer. 1998.
Mutational analysis of the Tup1 general repressor of yeast.Genetics
148:637-644.
5. Chen,
D., W. M. Toone, J. Mata, R. Lyne, G. Burns, K. Kivinen, A.
Brazma, N. Jones, and J. Bahler. 2003. Global
transcriptional responses of fission yeast to environmental stress.Mol. Biol. Cell
14:214-229.
6. Chen,
G., J. Fernandez, S. Mische, and A. J. Courey.1999
. A functional interaction between the histone
deacetylase Rpd3 and the corepressor groucho in Drosophila
development. Genes Dev.
13:2218-2230.
7. Courey,
A. J., and S. Jia. 2001. Transcriptional
repression: the long and the short of it. Genes Dev.
15:2786-2796.
8. Davie,
J. K., D. G. Edmondson, C. B. Coco, and
S. Y. Dent. 2003. Tup1-Ssn6 interacts with
multiple class I histone deacetylases in vivo. J.
Biol. Chem.
278:50158-50162.
9. Davie,
J. K., R. J. Trumbly, and S. Y. Dent.2002
. Histone-dependent association of Tup1-Ssn6 with
repressed genes in vivo. Mol. Cell. Biol.
22:693-703.
10. Edmondson,
D. G., M. M. Smith, and S. Y. Roth.1996
. Repression domain of the yeast global repressor Tup1
interacts directly with histones H3 and H4. Genes Dev.
10:1247-1259.
11. Ekwall, K. 2004. The RITS complex-A direct link between small RNA and heterochromatin. Mol. Cell 13:304-305.[CrossRef][Medline]
12. Greenall,
A., A. P. Hadcroft, P. Malakasi, N. Jones, B. A.
Morgan, C. S. Hoffman, and S. K.
Whitehall. 2002. Role of fission yeast Tup1-like
repressors and Prr1 transcription factor in response to salt stress.Mol. Biol. Cell
13:2977-2989.
13. Hirota,
K., T. Hasemi, T. Yamada, K. I. Mizuno, C. S.
Hoffman, T. Shibata, and K. Ohta. 2004. Fission yeast
global repressors regulate the specificity of chromatin alteration in
response to distinct environmental stresses. Nucleic Acids
Res.
32:855-862.
14. Hirota,
K., C. S. Hoffman, T. Shibata, and K. Ohta.2003
. Fission yeast Tup1-like repressors repress chromatin
remodeling at the fbp1+ promoter and the ade6-M26 recombination
hotspot. Genetics
165:505-515.
15. Huang, M., Z. Zhou, and S. J. Elledge. 1998. The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell 94:595-605.[CrossRef][Medline]
16. Janoo,
R. T., L. A. Neely, B. R. Braun,
S. K. Whitehall, and C. S. Hoffman.2001
. Transcriptional regulators of the
Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like
corepressors and the CCAAT binding factor activation complex.Genetics
157:1205-1215.
17. Jia,
S., K. Noma, and S. I. Grewal. 2004.
RNAi-independent heterochromatin nucleation by the stress-activated
ATF/CREB family proteins. Science
304:1971-1976.
18. Kim, H. S., E. S. Choi, J. A. Shin, Y. K. Jang, and S. D. Park. 2004. Regulation of Swi6/HP1-dependent heterochromatin assembly by cooperation of components of the MAP kinase pathway and a histone deacetylase Clr6. J. Biol. Chem.
19. Komachi,
K., and A. D. Johnson. 1997. Residues in the
WD repeats of Tup1 required for interaction with 2.Mol. Cell. Biol.
17:6023-6028.[Abstract]
20. Kondrashov, F. A., I. B. Rogozin, Y. I. Wolf, and E. V. Koonin. 2002. Selection in the evolution of gene duplications.Genome Biol. 3:RESEARCH0008.
21. Lee,
M., S. Chatterjee, and K. Struhl. 2000. Genetic
analysis of the role of Pol II holoenzyme components in repression by
the Cyc8-Tup1 corepressor in yeast. Genetics
155:1535-1542.
22. Mennella,
T. A., L. G. Klinkenberg, and R. S.
Zitomer. 2003. Recruitment of Tup1-Ssn6 by yeast
hypoxic genes and chromatin-independent exclusion of TATA binding
protein. Eukaryot. Cell
2:1288-1303.
23. Mukai,
Y., J. K. Davie, and S. Y. Dent.2003
. Physical and functional interaction of the yeast
corepressor Tup1 with mRNA 5'-triphosphatase.J. Biol. Chem.
278:18895-18901.
24. Mukai,
Y., E. Matsuo, S. Y. Roth, and S. Harashima.1999
. Conservation of histone binding and transcriptional
repressor functions in a Schizosaccharomyces pombe Tup1p
homolog. Mol. Cell. Biol.
19:8461-8468.
25. Papamichos-Chronakis,
M., R. S. Conlan, N. Gounalaki, T. Copf, and D.
Tzamarias. 2000. Hrs1/Med3 is a Cyc8-Tup1 corepressor
target in the RNA polymerase II holoenzyme. J. Biol.
Chem.
275:8397-8403.
26. Papamichos-Chronakis, M., T. Petrakis, E. Ktistaki, I. Topalidou, and D. Tzamarias.2002 . Cti6, a PHD domain protein, bridges the Cyc8-Tup1 corepressor and the SAGA coactivator to overcome repression at GAL1.Mol. Cell 9:1297-1305.[CrossRef][Medline]
27. Pelletier,
B., J. Beaudoin, C. C. Philpott, and S. Labbe.2003
. Fep1 represses expression of the fission yeast
Schizosaccharomyces pombe siderophore-iron transport system.Nucleic Acids Res.
31:4332-4344.
28. Proft, M., A. Pascual-Ahuir, E. de Nadal, J. Arino, R. Serrano, and F. Posas. 2001. Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress.EMBO J. 20:1123-1133.[CrossRef][Medline]
29. Provost,
P., R. A. Silverstein, D. Dishart, J. Walfridsson, I.
Djupedal, B. Kniola, A. Wright, B. Samuelsson, O. Radmark, and K.
Ekwall. 2002. Dicer is required for chromosome
segregation and gene silencing in fission yeast cells. Proc.
Natl. Acad. Sci. USA
99:16648-16653.
30. Redd,
M. J., M. B. Arnaud, and A. D.
Johnson. 1997. A complex composed of Tup1 and Ssn6
represses transcription in vitro. J. Biol.
Chem.
272:11193-11197.
31. Robyr, D., Y. Suka, I. Xenarios, S. K. Kurdistani, A. Wang, N. Suka, and M. Grunstein. 2002. Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases.Cell 109:437-446.[CrossRef][Medline]
32. Smith, R. L., and A. D. Johnson. 2000. Turning genes off by Ssn6-Tup1: a conserved system of transcriptional repression in eukaryotes. Trends Biochem. Sci. 25:325-330.[CrossRef][Medline]
33. Sprague, E. R., M. J. Redd, A. D. Johnson, and C. Wolberger. 2000. Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast. EMBO J. 19:3016-3027.[CrossRef][Medline]
34. Taricani,
L., H. E. Feilotter, C. Weaver, and P. G.
Young. 2001. Expression of hsp16 in response to
nucleotide depletion is regulated via the spc1 MAPK pathway in
Schizosaccharomyces pombe. Nucleic Acids Res.
29:3030-3040.
35. Treitel,
M. A., and M. Carlson. 1995. Repression by
SSN6-TUP1 is directed by MIG1, a repressor/activator protein.Proc. Natl. Acad. Sci. USA
92:3132-3136.
36. Tzamarias,
D., and K. Struhl. 1995. Distinct TPR motifs of Cyc8
are involved in recruiting the Cyc8-Tup1 corepressor complex to
differentially regulated promoters. Genes Dev.
9:821-831.
37. Tzamarias, D., and K. Struhl. 1994. Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex.Nature 369:758-761.[CrossRef][Medline]
38. Varanasi, U. S., M. Klis, P. B. Mikesell, and R. J. Trumbly. 1996. The Cyc8 (Ssn6)-Tup1 corepressor complex is composed of one Cyc8 and four Tup1 subunits. Mol. Cell. Biol. 16:6707-6714.[Abstract]
39. Watson,
A. D., D. G. Edmondson, J. R. Bone, Y.
Mukai, Y. Yu, W. Du, D. J. Stillman, and S. Y.
Roth. 2000. Ssn6-Tup1 interacts with class I histone
deacetylases required for repression. Genes Dev.
14:2737-2744.
40. Verdel,
A., S. Jia, S. Gerber, T. Sugiyama, S. Gygi, S. I. Grewal,
and D. Moazed. 2004. RNAi-mediated targeting of
heterochromatin by the RITS complex. Science
303:672-676.
41. Wu, J., N. Suka, M. Carlson, and M. Grunstein. 2001. TUP1 utilizes histone H3/H2B-specific HDA1 deacetylase to repress gene activity in yeast. Mol. Cell 7:117-126.[CrossRef][Medline]
42. Xue, Y., L. B. S. A. Haas, A. Gusnanto, M. Reimers, D. Talibi, M. Vingron, K. Ekwall, and A. P. H. Wright. 2004. A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift. Yeast 21:25-39.[CrossRef][Medline]
43. Zhang, Z., U. Varanasi, P. Carrico, and R. J. Trumbly.2002 . Mutations of the WD repeats that compromise Tup1 repression function maintain structural integrity of the WD domain trypsin-resistant core. Arch. Biochem. Biophys. 406:47-54.[CrossRef][Medline]
44. Zhang,
Z., U. Varanasi, and R. J. Trumbly. 2002.
Functional dissection of the global repressor Tup1 in yeast: dominant
role of the C-terminal repression domain. Genetics
161:957-969.
45. Znaidi, S., B. Pelletier, Y. Mukai, and S. Labbe. 2003. The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1. J. Biol. Chem. 279:9462-9474.
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||
2-fold out of 4,905 genes totally.
(B) Data generated under stress induction for 45 min (YES
plus 1 M KCl at 30°C). A total of 29 genes (red)were affected 
