From the Research and Education Center for Genetic
Information, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, § DNA Chip Research, Inc.
1-1-43 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, and
¶ Division of Molecular and Cellular Biology, Graduate School of
Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
Received for publication, December 27, 2002, and in revised form, January 28, 2003
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ABSTRACT |
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In Saccharomyces cerevisiae,
two highly conserved proteins, Rvb1p/Tih1p and Rvb2p/Tih2p, have been
demonstrated to be major components of the chromatin-remodeling INO80
complex. The mammalian orthologues of these two proteins have been
shown to physically associate with the TATA-binding protein (TBP)
in vitro but not clearly in vivo. Here we show
that yeast proteins interact with TBP under both conditions. To assess
the functional importance of these interactions, we examined the effect
of mutating both TIH2/RVB2 and SPT15, which
encodes TBP, on yeast cell growth. Intriguingly, only those
spt15 mutations that affected the ability of TBP to bind to
the TATA box caused synthetic growth defects in a
tih2-ts160 background. This suggests that Tih2p might be important in recruiting TBP to the promoter. A DNA microarray technique
was used to identify genes differentially expressed in the
tih2-ts160 strain grown at the restrictive temperature. Only 34 genes were significantly and reproducibly affected; some up-regulated and others down-regulated. We compared the transcription of several of these Tih2p target genes in both wild type and various mutant backgrounds. We found that the transcription of some genes depends on functions possessed by both Tih2p and TBP and that these
functions are substantially impaired in the
spt15/tih2-ts160 double mutants that confer
synthetic growth defects.
Nucleosomes are the primary components of chromatin, and their
structure and position can play a crucial role in determining eukaryotic transcription (1, 2). Certain structural configurations of
chromatin have been shown to repress transcription by blocking the
access of the transcriptional apparatus to its target promoters (1-4).
Conversely, DNA unwinding and re-configuration of nucleosomal structures are thought to allow transcription factors to bind to their
sites, thereby facilitating transcription initiation (1-4). Various
factors and multiprotein complexes have been identified that regulate
transcription by remodeling the structure of the nucleosome, and some
of these have been shown to participate crucially in the
transcriptional regulation of particular sets of genes (3, 4). These
factors and complexes require ATPase activity and/or function by
covalently modifying histones through acetylation, methylation, or
phosphorylation (4-6). Alteration of chromatin structure is an
important step not only for transcriptional regulation but also for DNA
repair, recombination, and replication (7, 8). Recent studies show that
individual chromatin-modifying complexes are apparently involved in
multiple DNA processing reactions (9-13). This includes the INO80
chromatin remodeling complex in Saccharomyces cerevisiae,
which appears to be involved in both transcription and DNA repair (9).
This complex contains the Ino80p ATPase, which belongs to the SWI2/SNF2
superfamily, and two ATP-dependent DNA helicases called
RuvB-like protein 1 (Rvb1p/Tih1p) and RuvB-like protein 2 (Rvb2p/Tih2p), which share homology with the prokaryotic DNA helicase
RuvB. RuvB acts on the process of branch migration at Holliday
junctions at the late stages of homologous recombination in DNA repair
(14, 15). Consistent with this, ino80 mutants are
susceptible to agents that cause DNA damage (9). The Tip60-containing
complex has been shown to contain the human orthologues of Rvb1p/Tih1p
and Rvb2p/Tih2p, which have also been designated as Tip60-associated
protein 54 A number of studies have demonstrated that Rvb1p/Tih1p and Rvb2p/Tih2p
play a crucial role in transcription. First, the rat orthologue of
Rvb1p/Tih1p was originally identified as a TATA-binding protein
(TBP)-interacting protein and was therefore denoted as TIP49a (16).
Second, the human orthologue of Rvb1p/Tih1p was shown to bind to the
RNA polymerase II holoenzyme complex (17). In this study, the
orthologue was denoted as RUVBL1 (RuvB-like protein 1). Notably, the mammalian orthologues of
Rvb1p/Tih1p and Rvb2p/Tih2p have been discovered independently many
times and consequently have a variety of designations, including
Pontin52/TIP49/TIP49a/RUVBL1/ECP-51/TAP54 A number of studies have suggested that Rvb1p/Tih1p and Rvb2p/Tih2p and
their mammalian orthologues possess diverse functions, in addition to
transcriptional regulation (we hereafter will refer to Rvb1p/Tih1p and
Rvb2p/Tih2p as Tih1p and Tih2p and their mammalian orthologues as
TIP49a and TIP49b, respectively). Indeed, these proteins appear to be
involved in various apparently distinct biological events, although
their precise functions are not completely understood (27-33). As a
first step to clarify the precise role of Tih1p and Tih2p in
transcription, we studied their functional interaction with TBP using
biochemical and genetic approaches. Here we show for the first time
that these proteins clearly form a complex with TBP in vivo.
Significantly, the combination of a temperature-sensitive mutation in
the TIH2 gene (tih2-ts160) with mutations in the
gene encoding TBP (SPT15) defective for TATA binding
resulted in synthetic growth defect. Some of the Tih2p target genes
that we identified by DNA microarray analyses using a
tih2-ts160 strains were also specifically affected in these
spt15 mutants. These observations strongly argue that the cooperative and functional interactions between Tih1p/Tih2p and TBP are
crucial for the transcription of at least a subset of yeast genes.
Yeast Strains, Media, and Plasmid
Constructions--
Yeast strains used in this report are listed
in Table I. Rich medium (YPD) and
synthetic complete (SD) medium with 5-fluoro-orotic acid (5-FOA) and
appropriate nutrients were prepared as described previously (34).
Standard methods were used to genetically manipulate yeast cells as
described previously (34, 35). Details of primers used in this study
are available on request. The TIH1 locus of genomic DNA was
amplified by PCR with primers that introduce
XhoI-NotI sites at each end. The
XhoI/NotI-digested 2-kb DNA fragment was then
ligated into the pRS316 and pRS314 vectors to obtain
pRS316-TIH1 and pRS314-TIH1, respectively.
Similarly, the EcoRI-NotI genomic DNA fragment
(2.3 kb) containing the TIH2 locus was amplified by PCR with
primers that create XhoI-NotI sites at each end.
The XhoI/NotI-digested 2.3-kb DNA fragment was
then ligated into pRS314 and pRS315 vectors to obtain
pRS314-TIH2 and pRS315-TIH2, respectively. Likewise, pRS315-tih2-ts160 was constructed by PCR with
primers that create XhoI-NotI sites at each
end.
Tih1p and Tih2p constructs with the native promoters were tagged with a
triple hemagglutinin (HA) epitope tag at their amino-terminal and
carboxyl-terminal ends, respectively, using fusion PCR methods (36).
Primers were designed to introduce EcoRI and
BamHI sites at the 5'- and 3'-ends of the triple HA-tag,
respectively. The final PCR products were subcloned as ~2.1- and
2.4-kb XhoI/NotI-digested fragments into the
pRS314 vector to generate pRS314-HA-TIH1 and pRS314-TIH2-HA, respectively. Similarly, TBP with a triple
FLAG epitope tag at its amino-terminal end was also constructed by the
fusion PCR method. The final PCR product was subcloned as a ~2.4-kb
EcoRI/NotI-digested fragment into the pRS313
vector to generate pRS313-FLAG-TBP. The TRP1-marked plasmid
carrying TBP or its derivatives and the hexahistidine-tagged
TBP expression vector for bacterial cells were described previously
(37). pGEX-3X-TIH1 and pGEX-3X-TIH2 plasmids were
constructed by inserting the TIH1 or TIH2 ORFs
amplified by PCR into the BamHI-EcoRI sites of
pGEX-3X (Amersham Biosciences).
The TIH1 knockout plasmid, pDisTIH1, was
constructed by PCR amplification of the 5'- and 3'-flanking DNA (~1
and 0.8 kb, respectively) of the TIH1 ORF, with the addition
of the appropriate restriction sites at each end, and insertion into
the pRS305 vector. To replace the entire TIH1 ORF with the
LEU2 gene on the genome, pDisTIH1 was linearized
with BamHI and transformed into a diploid strain (FY strain)
(25). Disruption was confirmed by Southern blotting. After
transformation of pRS316-TIH1 into this strain, the
Ura+ Leu+ haploid strain was selected by tetrad
analysis as the parental strain for plasmid shuffling. The YHO1 and
YHO2 strains were subsequently obtained from this parental strain by
plasmid shuffling (Table I). The YHO4 and YHO5 strains were obtained as
follows. A Ura+ Leu+ His
To obtain the parental strain containing double deletions of the
chromosomal TIH2 and SPT15 genes, for subsequent
plasmid shuffling, a diploid strain was created that had a heterozygous disruption of the TIH2 gene by insertion of the
HIS3 gene. This strain was transformed with the
hisG cassette plasmid for SPT15 gene disruption
(37). Disruption was confirmed by Southern blotting after removing the
URA3 gene situated in between the hisG sequences by growth on 5-FOA plates. This strain therefore bears heterozygous disruptions of the TIH2 and SPT15 genes and was
further transformed with URA3 and LYS2-marked
plasmids carrying the SPT15 and TIH2 genes,
respectively, and subsequently sporulated and dissected to obtain
haploid strains containing double deletions of the chromosomal TIH2 and SPT15 genes. Most of the other strains
listed in Table I were obtained from this parental strain by plasmid
shuffling using 5-FOA and Whole Cell Extract Preparation and
Immunoprecipitation--
Procedures were based on methods described
previously (25) with several modifications. Yeast strains incubated in
20 ml YPD at 30 °C (A600 = 1) were washed
once with lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA) and resuspended in 200 µl of lysis buffer with
protease inhibitors (10 µg/ml each of aprotinin, leupeptin, pepstatin
A, and 1 mM phenylmethylsulfonyl fluoride), and cell lysates were extracted by glass beads. The lysates (175 µl) were preincubated for 30 min at 4 °C in four volumes of
immunoprecipitation buffer (1.25% Triton X-100, 180 mM
NaCl, 6 mM EDTA, 60 mM Tris-HCl, pH 8.0, 6%
skim milk) containing 10 µl of protein A-Sepharose beads. The
supernatants were collected, and 0.5 µl of anti-TBP polyclonal
antibodies or preimmune antibodies were added and incubated on a
rotator overnight at 4 °C. 15 µl of protein A-Sepharose was then
added, followed by incubation for 1 h at 4 °C. The beads were
then washed three times with wash buffer (50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl, 5 mM
EDTA), and 25 µl of SDS sampling buffer was added. The samples were
boiled for 5 min and fractionated on 10% (for HA-Tih1p and HA-Tih2p)
or 12% (for TBP) SDS-polyacrylamide gels and then blotted onto
nitrocellulose membranes. HA-Tih1p/Tih2p and TBP were detected with the
monoclonal anti-HA 12CA5 antibody (Roche Molecular Biochemicals)
and the polyclonal anti-TBP antibody, respectively. For the reciprocal immunoprecipitation analysis, the monoclonal anti-HA 12CA5 antibody was
used, and detection was performed with the monoclonal anti-HA 12CA5 and
anti-FLAG antibodies (Sigma).
Preparation of Recombinant Proteins and GST Pulldown
Assay--
Hexahistidine-tagged TBP was expressed in Escherichia
coli BL21-codonPlus, (DE3)-RIL (Stratagene) by incubation with 0.1 mM isopropyl-1-thio- Synthetic Lethal Assay--
YHOTM1-13 and their wild type
strains (Table I) were grown in liquid YPD medium at 23 °C to the
early log phase and then spotted on 5-FOA plates and incubated at
29 °C for an additional 5 days. Likewise, YHOTW and YHOTD1-YHOTD3
strains were grown in liquid YPD medium to the early log phase at
23 °C and then spotted on YPD plates and incubated at 32 °C for
an additional 4 days.
Microarray Analysis--
Wild type (CRH6) and
thi2-ts160 (CRH8) strains were grown in YPD medium at
23 °C to an A600 of ~0.4. Preheat shock
treatment was performed by incubating the cells at 34 °C for 15 min
(38). Cells were incubated at 23 °C for 45 min and then shifted to
34 °C for an additional 45 min of incubation before being harvested. Total RNA was extracted by the hot phenol method (25), and
poly(A)+ RNA was prepared by using an oligo(dT) column
(Amersham Biosciences). Microarray analysis was performed as described
in Ref. 39 using Yeast Chip, version 1.0 (Hitachi Software
Engineering, Yokohama, Japan).2 Briefly,
fluorescently labeled cDNA was made from 1 µg of
poly(A)+ RNA in a reaction volume of 20 µl containing 0.5 µg of oligo-(dT) 18-mer, 2 µl of a dNTP mixture mix (consisted of 5 mM each of dATP, dCTP, and dGTP and 2 mM dTTP),
2 µl of 1 mM Cy3- or Cy5-conjugated dUTP, and 400 units
of Superscript II reverse transcriptase (Invitrogen). The two labeled
cDNA pools were combined and competitively hybridized to a
microarray under a coverslip for 16 h at 65 °C. Experiments were done in duplicate using different batches of template mRNA prepared under the same experimental condition. Fluorescent array images were obtained for Cy3 and Cy5 emissions by using a ScanArray Lite (PerkinElmer Life Sciences) scanner. Image intensity data were analyzed by using QuantArray 3.0 (PerkinElmer Life Sciences) software. Statistical analyses were carried out using Microsoft EXCEL.
Spots that have mean intensity values of less than 1000 arbitrary units
were discarded. Expression ratio (Cy3 (or Cy5) signal intensity/Cy5 (or
Cy3) signal intensity) was calculated and expressed as a logarithmic
value. The average from duplicated experiments was taken as the final
expression ratio. Highly irreproducible data, as judged from S.D., were discarded.
RNA Preparation and Northern Blot Analysis--
RNA preparation
and Northern blot analysis were performed as described (25). Strains
grown in YPD medium at 23 °C to the early exponential phase
(A600, ~0.2) were shifted to 34 °C and harvested at the indicated time. 10 µg of each RNA was subjected to
electrophoresis in a 1% formaldehyde/agarose gel, followed by transfer
to a nylon membrane. All probes used for hybridization were generated
by PCR amplification using genomic DNA as template and labeled using a
random primer labeling kit (TaKaRa).
Tih1p and Tih2p Interact with TBP in Vivo--
TIP49a was
originally purified as a TBP-interacting protein from rat nuclear
extract in vitro (16). More recently, GST pulldown experiments showed that both TIP49a and TIP49b proteins could directly
bind to TBP (18, 19). Nonetheless, the in vivo interaction between TIP49a/TIP49b and TBP had not been clearly established, as
their interaction in the rat nuclear extracts appears to be quite weak
(16), and TBP has consistently failed to be identified as a component
in several TIP49a/TIP49b-containing complexes (10, 21, 23). To examine
whether Tih1p and Tih2p, the yeast orthologues of TIP49a and TIP49b,
respectively, could complex with TBP in vivo,
immunoprecipitation (IP) analyses were performed using cell lysates prepared from yeast strains expressing Tih1p tagged at the
amino terminus or Tih2p tagged at the carboxyl terminus.
Immunoprecipitates obtained with the polyclonal anti-TBP antibody or
preimmune antiserum were subsequently probed with the anti-HA antibody
(Fig. 1A, upper panel). Both Tih1p and Tih2p proteins were found to be
specifically coprecipitated with endogenous TBP (Fig. 1A,
compare upper and lower panels). The same results
were obtained irrespective of the position or type of epitope tag used
(data not shown). To further confirm the interaction between
Tih1p/Tih2p and TBP, we conducted reciprocal IP experiments (Fig.
1B). Lysates prepared from yeast strains expressing
FLAG-tagged TBP and either HA-tagged Tih1p or Tih2p were precipitated
with anti-HA antibody and subsequently probed with anti-FLAG antibody
(Fig. 1B, lower panel). A single specific
immunoreactive band corresponding to FLAG-tagged TBP was detected on
the blot of samples from cells coexpressing HA-tagged Tih1p or Tih2p
(Fig. 1B, lanes 4 and 8). These
observations indicate that at least some of the Tih1p/Tih2p and TBP
molecules form a complex in yeast cells.
Tih1p and Tih2p Interact Directly with TBP in Vitro--
Previous
studies showed that mammalian orthologues of Tih1p/Tih2p could bind to
TBP directly (18, 19). We therefore conducted GST pulldown experiments
to examine whether yeast Tih1p/Tih2p could also bind directly to TBP
in vitro (Fig. 2). Recombinant TBP was mixed with the same amounts of GST-Tih1p, GST-Tih2p, or GST
alone (Fig. 2, lower panel). After incubation, complexes
bound to the glutathione-Sepharose 4B beads were analyzed by Western blotting with anti-TBP antibody. The bands corresponding to TBP were
detected only when recombinant TBP was mixed with GST-Tih1p or Tih2p
(Fig. 2, upper panel). Thus, yeast Tih1p and Tih2p can bind
to TBP directly.
TIH2 and SPT15, Encoding TBP, Interact Genetically--
Our
observation that Tih1p and Tih2p can interact with TBP both in
vitro and in vivo implies that these proteins may
function in transcriptional regulation as a single entity. In this
respect, it is intriguing that the temperature-sensitive
tih2-ts160 mutant we isolated previously (25) exhibited
phenotypes similar to those of taf1 mutants (25). The
TAF1 gene encodes a TFIID subunit that directly associates
with TBP (40). Both mutants were defective in the transcription of
genes encoding the ribosomal proteins and G1 cyclins and
exhibited a G1 cell cycle arrest phenotype (25, 41-43). In
addition, an allele of taf1 that lacks the TBP binding
domain acts as a synthetic lethal in the presence of various mutants of
spt15, the gene encoding TBP (37). Interestingly, these TBP
mutants were defective specifically in the post-recruitment step of TBP
to the core promoter (37). Hence, we reasoned that the
tih2-ts160 allele might also exhibit synthetic growth
defects when combined with various mutants of spt15.
We constructed yeast strains lacking both the TIH2 and
SPT15 genes and containing a URA3-marked plasmid
encoding wild type TBP and a LEU2-marked plasmid encoding
either the wild type TIH2 gene or the tih2-ts160
gene. These strains were subsequently transfected with
TRP1-marked plasmids encoding wild type TBP or with mutant TBPs that are defective in various TBP functions (37, 44-47). The
location of the mutation site of each TBP derivative is
indicated on TBP crystal structure shown in Fig.
3A (48). The synthetic growth
defects resulting from the combined expression of these mutant TBPs and
the tih2-ts160 allele was assessed by examining growth rates
on 5-FOA plates at 29 °C (Fig. 3B). We observed severe synthetic growth defects with the TBP-V161A and TBP-N159D mutants, whereas TBP-N159L and TBP-S118L showed weak but discernible defects (Fig. 3B). Intriguingly, all of these TBP mutants are
impaired in their ability to bind to the TATA element (44, 47). We observed no synthetic growth defects with any of the remaining TBP
mutants (Fig. 3B), which are apparently impaired in other functions (37, 47). This suggests that the TATA binding activity of TBP
is absolutely essential for yeast cell growth when TIH2 function is
impaired. Note that these synthetic growth defects were not observed at
lower temperatures (e.g. at 23 °C; data not shown),
possibly because TATA binding activity is partially restored. In
addition, on YPD plates, only those spt15 alleles having the strongest phenotypes, such as spt15-V161A and
spt15-N159D, exhibited synthetic growth defects (Fig.
3C). This suggests that rich medium may lower the
requirement for TATA binding activity of TBP in cell growth and
transcriptional regulation. Similar defects were observed when yeast
strains were cultured in liquid YPD media (Fig. 3D).
Thus, different synthetic phenotypes are observed when spt15
mutations are combined with either taf1 mutant alleles
lacking the TBP binding function (37) or with tih2 mutants.
This suggests that distinct functions of TBP are essential for cell
growth depending on which gene, i.e. TAF1 or
TIH2, is mutated.
Genome-wide Analysis of Gene Expression in the tih2-ts160
Mutant--
Both Tih1p and Tih2p participate in a chromatin-remodeling
complex composed of about 12 distinct polypeptides, including Ino80p, a
SWI2/SNF2 superfamily protein (9). However, like their mammalian orthologues (10, 22, 23), Tih1p and Tih2p may also be part of other
distinct multiprotein complexes, as suggested by Wu and co-workers (9).
Supporting this idea is the observation that the TIH1 and
TIH2 genes are essential for yeast cell growth, whereas the
INO80 gene is not (9). Furthermore, in a study of
genome-wide gene expression, Dutta and co-workers (26) found that
depletion of Tih1p or Tih2p using the temperature-inducible N-degron
system affected expression of about 5% of yeast genes in cells grown in galactose-containing media. This system should result in the total
degradation of the target proteins and thus should disrupt any complex
containing Tih1p or Tih2p.
The tih2-ts160 allele affects the function but not the
stability of the TIH2 protein (25). In good agreement with this notion, the G1 cell cycle arrest phenotype observed with the
tih2-ts160 allele is completely reversible (25). Thus, we
reasoned that if this mutant is shifted to the restrictive temperature,
we might observe a different genome-wide pattern of gene expression
profile from that obtained by Dutta and co-workers (26). We chose to cultivate the yeast strains in YPD medium, containing glucose, as this
would allow us to compare our results with those of other genome-wide
studies (49-51), which have mostly been performed in YPD. We hoped
that identification of the target genes for Tih2p by DNA microarray
experiments would allow the comparison of their expression profiles in
various mutants and help to elucidate some Tih2p functions. For
instance, we might be able to determine why synthetic growth defects
arise from a particular set of TBP mutants when combined with mutant
Tih2p. It would also be valuable to investigate how broadly the target
genes of Tih2p overlap with those of Ino80p.
To address these fundamental questions, we conducted DNA microarray
experiments using RNA prepared from wild type and tih2-ts160 mutant strains 45 min after shifting the temperature from 23 to 34 °C. Each RNA preparation was labeled with Cy3 or Cy5 fluorescent dye and then hybridized to a slide glass on which partial DNA fragments
corresponding to about 6000 different genes had been spotted (see
"Experimental Procedures"). Only 34 genes were significantly and
reproducibly affected (i.e. changed more than 2.5-fold) in the tih2-ts160 mutant at the restrictive temperature (see
Table II and Supplemental
Material). Of these, 20 genes showed a decrease in expression,
and 14 genes showed an increase. It is noteworthy that transcription of
the PHO and VTC genes, which are involved in
phosphate metabolism, decreased markedly in this mutant. Some of these
genes had been identified previously (9, 49-52) to be targets of the
SAGA, TFIID, SWI/SNF, NuA4, and INO80 complexes. Thus, the expression
of at least some of these PHO and VTC genes appear to require the integral function of multiple histone
acetyltransferase and chromatin remodeling complexes. In addition, only
two of the target genes we identified here were also identified by
Jonsson et al. (26), viz. MEP2 and
DLD3 (26). Among the most significantly affected genes, no
other genes were represented on both lists. Although the exact number
of overlapping target genes between the two studies is not certain,
they presumably correspond to target genes that are regulated by Tih2p
irrespective of the carbon source in the media.
Comparison of the Transcriptional Defects of the tih2-ts160 and
ino80 Null Mutants--
As mentioned, although Tih1p/Tih2p and Ino80p
are components of the INO80 complex, it is conceivable that these
proteins also function in other multiprotein complexes. If this is the
case, the transcriptional defects resulting from mutations in these factors might differ. To explore this possibility, the transcription of
several Tih2p target genes in wild type, tih2-ts160, and
ino80 null mutant strains were compared by Northern blotting
(Fig. 4). PHM2,
SPL2, PHO5, and MAE1 were selected as
representatives of genes showing decreased transcription in the
tih2-ts160 mutant (Fig. 4A), whereas
MEP2, GDH1, and DLD3 were selected as
genes showing increased transcription (Fig. 4B). As a
control, the transcription of the ADH1 and ACT1
genes, which are not affected by the tih2-ts160 allele, were
also examined. Quantification of each band is shown below the blot as
the ratio relative to the amount of mRNA recovered from the wild
type strain at the time of the temperature shift. Intriguingly, all of
the genes found to decrease in the tih2-ts160 background
also decreased in the ino80 null mutant. In contrast, MEP2 and GDH1 did not increase at all in the
ino80 null mutant, although DLD3 increased
slightly. These results indicate that the target genes of Tih2p and
Ino80p overlap but are not entirely identical. However, further
analysis is needed to determine whether the differential effects of
Tih2p and Ino80p on the transcription of the MEP2 and
GDH1 genes are because of functionally different defects of
a single complex (e.g. the INO80 complex) or defects in two
or more separate but as yet unidentified complexes.
Transcription of Tih2p Target Genes Was Specifically Affected in
the spt15 Mutants That Display Severe Synthetic Growth Defects When
Combined with tih2-ts160--
To investigate why the TBP mutants that
lack TATA binding activity show synthetic growth defects when combined
with the tih2-ts160 allele, we used Northern blotting to
measure the transcription of several Tih2p target genes in various
spt15 mutants after shifting the temperature from 23 to
34 °C (Fig. 5). We found earlier that although temperature shift (i.e. 34 °C) restricted the
growth of the tih2-ts160 mutant, they did not affect the
growth of any of the TBP mutants we tested here. However, combination
of some of the TBP mutants with tih2-ts160 were lethal even
at the permissive temperature (Fig. 3). It is possible that these TBP
mutants are impaired in functions that are shared with Tih2p and
defective in tih2-ts160. To investigate this possibility we
examined transcription of the SPL2 and PHO84
genes, both of which are Tih2p target genes whose transcription is
severely reduced in the tih2-ts160 strain. We found that
transcription of both genes were almost abolished in the
spt15-V161A mutant, as well (Fig. 5A). In
addition, transcription of the PHO5 gene, which was
decreased in the tih2-ts160 mutant, albeit to a lesser
extent than SPL2 and PHO84, was also reduced in
the spt15-V161A mutant (Fig. 5A). In contrast,
the MEP2 and GDH1 genes, whose expression
increased in tih2-ts160, was unchanged in the
spt15-V161A mutant (Fig. 5A). Additional
transcription defects were not observed when these two mutations were
combined (V161A x ts160; see Fig. 5A).
Taken together, these observations suggest that Tih2p can have both
stimulatory and inhibitory effects on transcription, both of which are
affected by the ts160 mutation, and that the stimulatory
function overlaps with that of the TBP and is impaired in the
spt15-V161A mutant.
Other TBP mutations that affect TATA binding also induced synthetic
growth defects when combined with the tih2-ts160 allele (Fig. 3). To examine the relationships among TATA binding,
transcription, and the consequent growth phenotypes of these mutant
alleles, we examined the transcription of five genes (PHM2,
SPL2, PHO5, GDH1, and ADH1)
in wild type strains versus strains harboring one of the
eleven spt15 mutant alleles, the tih2-ts160
allele, or both. Gene expression was analyzed 45 min after shifting to the restrictive temperature (Fig. 5B). Results of the
mRNA quantification are summarized in Fig. 5C. The
spt15-V161A and spt15-N159D alleles exhibited the
most severe synthetic growth defects when combined with the
tih2-ts160 allele (Fig. 3). In parallel, transcription of
the PHM2, SPL2, and PHO5 genes, which
are decreased in tih2-ts160 mutants, were almost abolished
in these two TBP mutants (Fig. 5, A and B). In
contrast, increased expression of the GDH1 gene was observed
only in the tih2-ts160 mutant (Fig. 5B). In the
spt15-N159D mutant, transcription of the GDH1
gene was considerably weaker even when the tih2-ts160
mutation was present. This suggests that N159D might impair TBP
function more strongly than other TBP mutations. Consistent with this
is the observation that even transcription of the ADH1 gene,
which showed no change in tih2-ts160 and was used as a
control, was reproducibly decreased in the spt15-N159D mutant (Fig. 5B).
Somewhat unexpectedly, the effect of the N159L mutation on
transcription was rather different from that of N159D (Fig.
5B). Although both showed synthetic growth defects when
combined with tih2-ts160 mutation and grown on synthetic
media (Fig. 3A), the N159L mutation alone uniquely increased
the transcription of genes like PHM2,
SPL2, and PHO5, whose transcription is decreased
in tih2-ts160 (Fig. 5C). This increase in
transcription was also observed in the double spt15-N159L,
tih2-ts160 mutant. Thus, the mechanisms causing synthetic
growth defects in the spt15-N159L and spt15-N159D
double mutants may differ even though they harbor substitutions at the
same residue. This may be responsible for the different growth
phenotypes of these two mutants on YPD media (Fig. 3B).
Another TBP mutant, S118L, which showed the weakest synthetic growth
defects (Fig. 3), also exhibited impaired transcription of the
PHM2 and SPL2 genes. Other TBP mutants that did
not display synthetic growth defects were almost normal in the
transcription of these genes, except for the increased expression of
GDH1 in the spt15-P65S mutant.
Thus, the extent to which TBP mutations cause a decrease in yeast
growth in the context of the tih2-ts160 mutation appears to
correlate with the decreased expression of several genes that are
independently affected by both mutations. We conclude that there are
functions of TBP and Tih2p that overlap and appear to be involved in
recruiting TBP to the promoter and that disrupting both of these
functions at the same time has a synthetic effect on yeast cell growth.
In this study, we verified that Tih2p and TBP physically and
functionally interact in vivo. Although it is unclear why
previous studies failed to show such a physical association in
vivo, it may be partly because of the relative weakness of their
interaction. In fact, we estimate that less than 1% of the total Tih2p
pool can be coprecipitated with TBP and vice versa.
Considering that other TBP-interacting partners, such as TBP-associated
factors, can be coprecipitated stoichiometrically with TBP, it
is likely that the interaction between Tih2p and TBP is transient
and/or consists of only a small fraction of these two proteins that can form a stable complex in vivo.
The most important findings presented in this report are that only some
spt15 alleles show a synthetic growth defect with the
tih2-ts160 allele. Furthermore, all mutation sites of these alleles locate on the surface side of DNA binding region of TBP, and
these TBP mutants all had defects in TATA binding. This suggests that
Tih2p might be involved in recruiting TBP and/or other TBP-related complexes such as TFIID to the promoter. Intriguingly, the integrity of
the TATA binding activity of TBP has been shown previously (44, 45) to
be important for the response to acidic activators like GAL4, and Tih1p
and Tih2p are also required for GAL4-dependent transcriptional activation (26). These observations are consistent with
a model in which TBP or related complexes are recruited by Tih1p and Tih2p.
We identified the target genes of Tih2p by DNA microarray analysis.
Comparison of severely affected genes in the tih2-ts160 strain with those identified previously (26) by depleting Tih1p and
Tih2p levels revealed that they did not perfectly overlap with each
other. The difference between these two studies might be because of
different experimental conditions, including the use of different
carbon sources, the application of the protein depletion method in the
Dutta study (26), and variations in the array-making technology used.
Importantly, increased expression of some target genes (e.g.
MEP2, GDH1, and DLD3) was observed in
the tih2-ts160 mutant but not in the ino80 null
mutant. This clearly indicates that Tih2p and Ino80p have distinct
functions in transcription, even though they are components of the same chromatin remodeling complex.
Notably, we found a correlation between the synthetic growth defects
and the expression profile of several genes (namely, PHM2,
SPL2, and PHO5); transcription of these genes was
abolished not only in the tih2-ts160 mutant but also in the
spt15-V161A and spt15-N159D mutants that
displayed the most severe synthetic growth defects. Thus, the same or
overlapping steps operating during the course of transcription, such as
the recruitment of TBP to the promoter, may be partially impaired by
both of these mutations, and combining them may result in
transcriptional defects in a much broader range of genes, thereby
leading to synthetic growth defects. Significantly, the effect of these
mutations on transcription seems to be independent of the physical
interaction between TBP and Tih2p, because immunoprecipitation assay
revealed that none of the TBP mutations appear to be defective in this interaction.3 Consistent with
this is the observation that overexpression of TBP did not rescue the
temperature-sensitive phenotype of the tih2-ts160
mutant.3 Therefore we speculate that the complex containing
both Tih proteins might change the neighboring chromatin environment in
some specific genes rather than by simply associating physically with
TBP. One of our possible working models of the function of Tih2p is
that the complex containing Tih1p and Tih2p might cause the chromatin remodeling in some specific genes through the interaction with acidic
activators and efficiently assist the recruitment of TBP to the TATA
box of these genes. According to this scenario, tih2-ts160 mutant might fail the activity of either chromatin remodeling of target
genes or interactions with acidic activators under the restrictive
temperature. We are currently conducting additional studies to examine
this possibility.
Our previous study demonstrated that at least some of the genes
affected in the taf1 mutant, e.g. cyclin and
ribosomal protein genes, were also affected in the
tih2-ts160 mutant. However, a recent study (53) using
proteomic approaches revealed that Taf14p is also a component of the
INO80 complex. This protein has also been shown to be a component of
multiple transcription complexes, including the TFIID, TFIIF, Swi-Snf,
NuA3, and mediator complexes (54-56). Furthermore, genes involved in
phosphate metabolism appeared to be a common target of the TFIID, SAGA,
Swi-Snf, NuA4, and INO80 complexes. These observations support the
notion that the INO80 complex may carry certain core promoter
functions, in addition to its chromatin-remodeling activity, such as
TFIID, TFIIF, and Mediater.
Tih1p and Tih2p both belong to the AAA+ family, a
superfamily of proteins designated the ATPases
associated with various cellular activities
family (57, 58), and may form hetero- or homohexameric ring-like
structures (9). Other members of this family are also known to play an
important role in transcription. For instance, a ring-like structure
that corresponds to part of the 19 S proteasome subcomplex can be
recruited to the GAL1 promoter by the GAL4 activator. This complex,
which is apparently composed of six AAA proteins (i.e.
Rpt1p-Rpt6p), was designated recently (59) as the APIS (AAA
proteins independent of 20 S)
complex. Biochemical and genetic studies demonstrated that the APIS
complex is essential for activation by GAL4 (59). However, it is
unclear how the AAA proteins in the APIS complex and Tih1p/Tih2p are
involved in transcription. Elucidation of the molecular defects
occurring in the tih2-ts160 mutant may bring us new insights
into the mechanisms of transcriptional regulation in eukaryotes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TAP54
)1 and
TAP54
, respectively (10). This human complex can bind to structural
DNA that mimics Holliday junctions. Importantly, ectopic expression of
a mutant Tip60 lacking its histone acetyltransferase activity
causes human cells to become defective in double-strand DNA break
repair (10). These observations indicate that the multiple roles of
individual chromatin-modifying complexes in various DNA processing
reactions may be evolutionarily conserved from yeast to man.
and
Reptin52/TIP48/TIP49b/RUVBL2/ECP-54/TAP54
, respectively. Third, both
of these proteins can bind to transcriptional regulatory factors,
e.g.
-catenin and c-Myc, and influence their function either positively or negatively (18-21). Furthermore, certain
TIP49-TIP48-BAF53-containing complexes were shown to be essential for
c-Myc-mediated transformation (21, 22). Although these three proteins
were originally identified as components of the Tip60-containing
complex described earlier (10), it is possible they can also function
distinct from Tip60, for example, as part of a p400 complex that is an
essential E1A transformation target (22, 23). Notably, BAF53 was also
found to be a component of the mammalian SWI/SNF-related
chromatin-remodeling complex. Fourth, TIP49 was recently shown to bind
to the E2F1 transactivation domain and to modulate its apoptotic
activities (24). Finally, we and others (25, 26) have demonstrated that
yeast Rvb1p/Tih1p and Rvb2p/Tih2p are required for the transcription of
at least a subset of genes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
haploid
was selected by tetrad analysis, and this strain was transformed with
pRS314-TIH1 or pRS314-HA-TIH1 and then grown on
5-FOA plates to remove pRS316-TIH1. These strains,
expressing TIH1 or HA-TIH1, were further successively transformed with
pRS313-FLAG-SPT15 and the hisG cassette plasmid
for SPT15 gene disruption (37). The URA3 gene
sandwiched by the two hisG sequences was removed by growth
on 5-FOA plates to generate the YHO4 and YHO5 strains, respectively.
Disruption of the SPT15 gene was confirmed by Southern blotting. The YHO3 strain, which was deleted for the TIH2
gene, was obtained from the parental CRPA1 strain (25) by plasmid shuffling in a manner similar to YHO1 and YHO2.
-aminoadipic acid to counterselected
against the URA3 and LYS2 genes, respectively.
The
ino80 strain is a generous gift from Carl Wu
(National Institutes of Health). All strains are of the S288C background.
-D-galactopyranoside for
4 h at 20 °C in 2 liters of 2× YT medium. The collected cells
were lysed by freeze-thawing and resuspended in 40 ml of
phosphate-buffered saline with 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml each of aprotinin, leupeptin, and pepstatin A, and
1% Triton X-100. After sonication and centrifugation, cell debris was
removed by filtration. The cell lysate was subjected to TALON metal
affinity resin (Clontech) to purify TBP. Control GST, GST-Tih1p, and GST-Tih2p were expressed in E. coli
BL21-codonPlus, (DE3)-RIL. After incubation with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside for 2 h at
25 °C in 500 ml of 2× YT medium, the cells were collected and
resuspended in 40 ml of phosphate-buffered saline with protease inhibitors and detergent and sonicated to collect the cell extracts as
described above. GST and GST fusion proteins were purified on
glutathione-Sepharose 4B (Amersham Biosciences). The GST pulldown assay
was performed as follows. 3 µg each of GST, GST-Tih1p, or GST-Tih2p
and 3 µg of TBP were incubated with a 10-µl bed volume of
glutathione-Sepharose in 1 ml of binding buffer (50 mM
Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA) for 1.5 h at 4 °C. Beads were washed
extensively with binding buffer and then boiled in 20 µl of SDS
sampling buffer. The eluted proteins were fractionated on 12%
SDS-polyacrylamide gel, blotted onto a nitrocellulose membrane, and
probed with the polyclonal anti-TBP antibody. Recombinant proteins were
quantified by the BCA protein assay kit (Pierce) and/or Coomassie
Brilliant Blue staining on the gel.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Tih1p and Tih2p interact with TBP in
vivo. A, whole cell extracts were prepared
from yeast strains expressing untagged Tih1p (lanes 1 and
4), untagged Tih2p (lanes 7 and 10),
HA-tagged Tih1p (lanes 2, 3, 5, and
6), or HA-tagged Tih2p (lanes 8, 9,
11, and 12). The expression of HA-Tih1p,
HA-Tih2p, and TBP was confirmed by immunoblotting with anti-HA and
anti-TBP polyclonal antibodies (lanes 1-3 and
7-9). 0.1 and 14% input proteins were loaded in the
upper and lower panels, respectively (lanes
1-3 and 7-9). Whole cell extract proteins were
immunoprecipitated with anti-TBP or preimmune antibodies as indicated
above the blot (lanes 4-6 and
10-12). Precipitates were fractionated by SDS-PAGE, blotted
onto the nitrocellulose membranes, and probed with the antibodies
indicated on the right side of the blot. B, for
the reciprocal IP experiments, whole cell extracts were prepared from
yeast strains expressing FLAG-tagged TBP (lanes 1-8),
together with untagged Tih1p (lanes 1 and 3),
untagged Tih2p (lanes 5 and 7), HA-tagged Tih1p
(lanes 2 and 4), or HA-tagged Tih2p (lanes
6 and 8). Precipitates with anti-HA or preimmune
antibodies were fractionated, blotted, and probed with the antibodies
indicated on the right side of the blot. 5.7 and 0.1% input
proteins were loaded in the upper and lower
panels, respectively (lanes 1 and 2 and
lanes 5 and 6).
View larger version (35K):
[in a new window]
Fig. 2.
Tih1p and Tih2p interact directly with TBP
in vitro. Recombinant GST, GST-Tih1p, and
GST-Tih2p (3 µg) were incubated with recombinant TBP (3 µg),
followed by precipitation with glutathione-Sepharose. Precipitated
proteins were fractionated by SDS-PAGE and immunoblotted with
polyclonal anti-TBP antibody (upper panel). 50% of input
GST and GST fusion proteins were fractionated by SDS-PAGE and stained
with Coomassie Brilliant Blue (CBB; lower panel).
The positions of the molecular size markers are indicated on
the left.
View larger version (27K):
[in a new window]
Fig. 3.
Genetic interaction between the
TIH2 and SPT15 genes. Mapping of
the each TBP derivatives on the crystal structure of
carboxyl-terminal domain of yeast TBP is shown. TBP has four helices (H1, H2, H1', and H2') and ten
strands (S1 to S5 and S1' to S5'). DNA
interacts with
strands on the concave surface of TBP (DNA
surface). B, TRP1-marked plasmids encoding
TBP or its derivatives as indicated on the left were
individually introduced into a strain lacking both the TIH2
and SPT15 genes and containing a URA3-marked
plasmid encoding wild type (WT) TBP, as well as with a
LEU2-marked plasmid encoding either the wild type
TIH2 gene or the tih2-ts160 gene, as indicated on
top. The resulting transformants were grown on 5-FOA plates
at 29 °C for 5 days. C, the transformants described for
A were grown on 5-FOA plates at 23 °C for several days to
remove the URA3-marked plasmid expressing wild type TBP. The
strains expressing wild type or mutant TBP from TRP1-marked
plasmids were then grown on YPD plates at 32 °C for 4 days.
D, growth curves of the wild type (closed
square), spt15-V161A mutant (open square),
tih2-ts160 mutant (closed circle), and
spt15-V161A/tih2-ts160 double mutant (open
circle) after shifting the temperature from 23 to 32 °C in YPD
liquid medium.
Genes affected by ts160 mutant
View larger version (66K):
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Fig. 4.
Comparison of transcription of several Tih2p
target genes in the wild type, tih2-ts160, and
ino80 null mutant strains. A, total
RNA was isolated from wild type (WT), tih2-ts160,
and ino80 strains at the indicated time after shifting
the temperature from 23 to 34 °C. 10 µg of total RNA prepared from
each strain was subjected to Northern blot analysis. DNAs corresponding
to the down-regulated genes in the tih2-ts160 mutant
indicated on the left were labeled as probes. The amount of
each transcript was quantified in duplicate. The average amount is
shown below the blot relative to the amount of mRNA
obtained from the wild type strain just at the point of the temperature
shift. One of the duplicate experiments is shown here as a blot.
B, the up-regulated genes in the tih2-ts160
mutant were examined as described for A.
View larger version (53K):
[in a new window]
Fig. 5.
Comparison of transcription of several Tih2p
target genes in the wild type, spt15,
tih2-ts160, and spt15/tih2-ts160
double mutant strains. A, total RNA was isolated
from the wild type (WT), spt15-V161A,
tih2-ts160, and spt15-V161A/tih2-ts160 double
mutant strains at the indicated time after shifting the temperature
from 23 to 34 °C. Northern blot analysis was conducted as described
for Fig. 4 with the probes indicated on the left. The amount
of each transcript was quantified as described for Fig. 4.
B, total RNA was isolated from strains whose
SPT15 and TIH2 alleles are indicated on the
left and the top, respectively, 45 min after
shifting the temperature from 23 to 34 °C. Northern blot analysis
was conducted as described for Fig. 4 with the probes indicated on the
bottom. C, summary of the results of mRNA
quantification obtained in B. The numbers on the
x axis represent the spt15 alleles as indicated
in B. Black and white bars represent
the wild type and mutant (ts160) alleles of the
TIH2 gene, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Carl Wu for gifts of yeast strains, Akiko Kobayashi for constructions of some TBP derivatives, Akiko Tsuda for protein preparations, Miki Matsumura for excellent technical assistance, Marc Lamphier for critical reading of the manuscript, and all the members of the Kohno laboratory for useful discussions of this work.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for scientific research and scientific research on priority areas (to K. K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.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.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental table.
To whom correspondence should be addressed. Tel.:
81-743-72-5640; Fax: 81-743-72-5649; E-mail:
kkouno@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M213220200
2 C. R. Lim, A. Fukakusa, and K. Matsubara, submitted for publication.
3 H. Ohdate and K. Kohno, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: TAP, Tip60-associated protein; TBP, TATA-binding protein; TIP, TBP-interacting protein; ORF, open reading frame; 5-FOA, 5-fluoro-orotic acid; HA, hemagglutinin; GST, glutathione S-transferase; IP, immunoprecipitation.
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REFERENCES |
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