From the Divisions of Molecular Genetics and
¶ Mutagenesis, Department of Molecular Genetics, National
Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
Received for publication, January 11, 2001, and in revised form, February 21, 2001
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ABSTRACT |
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The general transcription factor IID consists of
the TATA-binding protein (TBP) and multiple TBP-associated factors
(TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy
suppressors of a temperature-sensitive mutation in the
ubiquitin-conjugating enzyme gene ubcP4+.
The ubcP4ts mutation causes cell cycle arrest in
mitosis, probably due to defects in ubiquitination mediated by the
anaphase-promoting complex/cyclosome. One multicopy suppressor
is the previously reported gene taf72+,
whereas the other is a previously unidentified gene named
taf73+. We show that the
taf73+ gene, like
taf72+, is essential for cell viability. The
taf72+ and taf73+ genes
encode proteins homologous to WD repeat-containing TAFs such as human
TAF100, Drosophila TAF80/85, and Saccharomyces
cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins
are present in the same complex with TBP and other TAFs and that TAF72,
but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell
cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9+. These
results suggest that TAF72 and TAF73 may regulate the expression of
genes involved in ubiquitin-dependent proteolysis during
mitosis. Our study thus provides evidence for a possible role of WD
repeat-containing TAFs in the expression of genes involved in
progression through the M phase of the cell cycle.
The general transcription factor
(TF)1 IID plays a critical
role in transcription initiation of protein-coding genes by RNA polymerase II. TFIID is a multiprotein complex comprising the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs),
which have been well conserved from yeast to humans (1, 2). TBP
specifically recognizes TATA elements, whereas certain TAFs directly
interact with initiator or downstream promoter elements. In addition to
a role in core promoter recognition, TAFs have been proposed to
function as targets of activators. Subsets of TAFs have also been found
in histone acetylase complexes distinct from TFIID (3, 4).
To assess the requirement of individual TAFs for transcription in
vivo, yeast (Saccharomyces cerevisiae) mutants have
been used (5-13). Interestingly, inactivation of some TAFs results in
cell cycle phenotypes. yTAF130/145 inactivation leads to G1 arrest, whereas inactivation of yTAF90 or yTAF150/TSM1 results in
G2/M arrest (5, 7). Genome-wide expression analyses have identified sets of genes whose expression depends on yTAF17, yTAF23/25, yTAF60, yTAF61/68, yTAF90, or yTAF130/145 (14, 15). For example, upon
inactivation of yTAF90, ~8% of all yeast genes show a significant decrease in expression.
yTAF90 (16, 17) and its human and Drosophila homologs,
hTAF100 (18-20) and dTAF80/85 (21, 22), respectively, all contain WD
repeats. The WD repeat is a conserved sequence motif usually ending
with Trp-Asp (WD), and WD repeat-containing proteins are implicated in
a wide variety of cellular functions (23). yTAF90 and hTAF100 are also
components of histone acetylase complexes distinct from TFIID such as
the Spt-Ada-Gcn5 acetyltransferase (SAGA) and TBP-free
TAFII-containing (TFTC) complexes (24, 25). In addition, an
hTAF100-related protein, PAF65 Ubiquitin-dependent proteolysis has been shown to play a
key role in progression through the cell cycle (27). A
ubiquitin-protein ligase complex known as the anaphase-promoting
complex or cyclosome (APC/C) promotes the metaphase-to-anaphase
transition and the exit from mitosis by mediating ubiquitination of
anaphase inhibitors and mitotic cyclins, leading to their destruction
by the 26 S proteasome (28). In the fission yeast
Schizosaccharomyces pombe, the ubiquitin-conjugating enzyme
UbcP4 seems to be involved in APC/C-mediated proteolysis (29). First,
depletion of UbcP4, like mutations in APC/C subunit genes such as
cut9+, blocks the initiation of anaphase.
Second, overexpression of UbcP4 suppresses a cut9 mutation.
Finally, among the family of ubiquitin-conjugating enzymes, UbcP4 is
most closely related to clam E2-C, Xenopus UBCx, and human
UbcH10, all of which are involved in ubiquitination of mitotic cyclins.
We report here the isolation of two related TAF genes,
taf72+ and taf73+, from
S. pombe as multicopy suppressors of a temperature-sensitive ubcP4 mutation. TAF72 and TAF73 proteins have homology to WD
repeat-containing TAFs such as hTAF100, dTAF80/85, and yTAF90. We show
that both TAF72 and TAF73 are associated with TBP and other TAFs,
whereas only TAF72 is associated with Gcn5, a putative histone
acetylase. We also show that taf72+ and
taf73+ suppress a mutation in the
cut9+ gene. These results suggest that TAF72 and
TAF73 may regulate the expression of genes involved in progression
through the M phase of the cell cycle.
Yeast Strains, Media, and Molecular Genetic Methods--
The
following S. pombe strains were used in this study: JY741
(h Isolation of a ubcP4ts Mutation--
An
XhoI-XbaI fragment containing the
ubcP4+ gene was cloned into the
SalI/XbaI site of pUC19 Isolation and Characterization of Multicopy Suppressors of a
ubcP4ts Mutation--
A ubcP4-140 strain was
transformed with an S. pombe genomic library constructed
with the multicopy plasmid pSP1 (34). Leu+ transformants
selected on EMM + Ade at 25 °C were replica-plated onto EMM + Ade
containing phloxine B, and the plates were incubated at 35 °C.
Plasmids were recovered from white colonies grown at 35 °C and used
to retransform the ubcP4-140 strain. Subcloning of inserts
from two plasmids (pSP1-7 and pSP1-19) resulted in 2.6-kb
SacI-HindIII and 2.2-kb
SalI-HindIII fragments capable of suppression
(see Fig. 3A). Sequencing followed by data base searches
using the BLAST program revealed that the former contained the
taf72+ gene (35) and the latter contained a
related but previously unidentified gene. This gene was named
taf73+ and analyzed further.
taf73+ cDNA was amplified by reverse
transcription-PCR, cloned into pGEM-T (Promega), and sequenced. A
3'-portion of taf73+ cDNA was also amplified
by 3'-rapid amplification of cDNA ends and sequenced.
Disruption of the taf73+ Gene--
A 3.0-kb genomic
DNA fragment containing the
taf73+ gene was amplified by PCR and cloned into
pGEM-T to generate pGEM-T(taf73). The entire vector sequence flanked by
5'- and 3'-noncoding sequences of taf73+ was
amplified from pGEM-T(taf73) by PCR, digested with XhoI and SmaI, and ligated with a 1.8-kb
XhoI-SmaI ura4+ fragment
to generate pGEM-T( Construction of S. pombe Strains Expressing Epitope-tagged TAF or
Gcn5--
To construct S. pombe strains expressing FLAG or
HA epitope-tagged TAF protein, DNA fragments that encode epitope-tagged
TAF were amplified by PCR using primers with overlapping extension (37)
and used to replace the chromosome segment by transplacement (38).
Strains expressing FLAG-tagged TAF72 or TAF73, in which a FLAG epitope
(DYKDDDDK) was inserted at the N terminus of the TAF72 or TAF73
protein, were constructed as follows. DNA fragments containing both a
5'-noncoding region and a 5'-portion of the coding sequence with the
FLAG sequence immediately after the initiation codon were amplified by
PCR and cloned into pBluescript SK (Stratagene) carrying a
ura4+ fragment. The resulting plasmids were
linearized at a unique restriction site within the
taf genes (AatII for
taf72+ and SpeI for
taf73+) (see Fig. 3A) and used to
transform strain JY741. Integration into the taf locus on
the chromosome results in a full-length taf gene with
the FLAG sequence and a 3'-truncated taf gene that are separated by the ura4+ plasmid sequence.
Correct integration was confirmed by PCR. Recombination that occurs
upstream of the FLAG sequence leaves only a FLAG-TAF gene on the
chromosome. 5-Fluoroorotic acid-resistant segregants were screened by
PCR for the presence of the FLAG sequence to obtain
tafFLAG strains. Similarly, an S. pombe strain expressing HA-tagged TAF73, in which three copies of
an HA epitope (YPYDVPDYA) were inserted at the N terminus of the TAF73
protein, was constructed using strain JY746. This
taf73HA strain was crossed with the wild-type,
taf72FLAG, and taf73FLAG
strains described above to construct diploid strains expressing HA-TAF73; HA-TAF73 and FLAG-TAF72; and HA-TAF73 and FLAG-TAF73, respectively.
S. pombe strains expressing HA-tagged Gcn5, in which three
copies of an HA epitope were inserted at the N terminus of the Gcn5
protein, were constructed using the wild-type,
taf72FLAG, and taf73FLAG
strains. A DNA fragment containing a 5'-noncoding region and a
5'-portion of the coding sequence with the triple HA sequence immediately after the initiation codon was cloned into the
ura4+ plasmid. Linearization with
NruI followed by integration into the S. pombe
chromosome resulted in strains expressing HA-Gcn5; HA-Gcn5 and
FLAG-TAF72; and HA-Gcn5 and FLAG-TAF73, respectively.
Preparation of S. pombe Whole-cell Extracts--
S.
pombe strains were grown to an A600 of 0.5 (1 × 107 cells/ml) in 200 ml of YE + AdeUra medium at
30 °C. Cells were harvested, washed with H2O,
transferred to a 2.0-ml tube, and frozen at Immunoprecipitation and Immunoblotting--
For
immunoprecipitation with anti-FLAG antibody, 300-500 µl of
whole-cell extract was mixed with 0.2 volume of a slurry of anti-FLAG
M2-agarose and incubated for 2-4 h at 4 °C on a rotating wheel. For
immunoprecipitation with anti-TBP antibody, 200 µl of whole-cell
extract was mixed with 10 µl of anti-S. pombe TBP serum or
preimmune serum and incubated for 1 h on ice, and then 40 µl of
a slurry of protein A-Sepharose (Amersham Pharmacia Biotech) was added
and incubated for 1 h at 4 °C on a rotating wheel. The beads
were washed three times with 1 ml of buffer A, resuspended in 1.5× SDS
gel loading buffer, and frozen at Antibodies--
Rabbit anti-S. pombe TBP,
anti-S. pombe TAF130, and anti-S. pombe PTR6
polyclonal antibodies were kindly provided by Tetsuro Kokubo (Nara
Institute of Science and Technology). Mouse anti-FLAG M2 monoclonal
antibody and anti-FLAG M2-agarose were purchased from Sigma, and mouse
anti-HA monoclonal antibody (16B12) was purchased from BAbCO.
Peroxidase-conjugated goat anti-rabbit IgG (Cappel) and
peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch
Laboratories) were used as secondary antibodies.
Nucleotide Sequence Accession Number--
The sequence of the
taf73+ gene has been submitted to the
DDBJ/EMBL/GenBankTM Data Bank under accession number
AB039954. In the course of this study, we noticed that the sequence of
a genomic DNA fragment containing the taf73+
gene was determined by the S. pombe Genome Sequencing
Project. Our taf73+ sequence is identical to the
sequence of the predicted gene SPBC15D4.14 on cosmid c15D4 (accession
number AL031349).
Isolation of Putative TAF Genes as Multicopy Suppressors of a
ubcP4ts Mutation--
Depletion of the UbcP4 protein, an
S. pombe ubiquitin-conjugating enzyme, blocks the initiation
of anaphase in mitosis, suggesting a role of UbcP4 in cell cycle
progression through mitosis (29). To confirm and extend this result, we
isolated a temperature-sensitive mutation in the
ubcP4+ gene (designated ubcP4-140 or
ubcP4ts) as described under "Experimental
Procedures." A ubcP4-140 strain showed a rapid cessation
of cell growth when the culture was shifted from 25 to 36 °C (Fig.
1A). After the shift to
36 °C, the following types of cells accumulated: metaphase-arrested
cells with condensed chromosomes, septated cells without chromosome
segregation, and cells undergoing cytokinesis without chromosome
segregation (a cut phenotype) (Fig. 1B). At
6 h after the shift, ~30% of the cells showed septation or
cytokinesis without chromosome segregation. Thus, the
ubcP4ts mutation seems to block the initiation of
anaphase, thereby causing uncoordinated mitosis where septation or
cytokinesis occurs without chromosome segregation. This phenotype
closely resembles those caused by mutations in the cut genes
that encode components of APC/C such as cut9-665 (30) and
cut4-533 (39). It is therefore most likely that UbcP4, in
conjunction with APC/C, functions in ubiquitination of proteins
required for progression through mitosis, including the anaphase
inhibitor (securin) Cut2 and the mitotic cyclin Cdc13 (40).
The ubcP4-140 mutation was used for isolation of multicopy
suppressors that enable growth at 35 °C (see "Experimental
Procedures") (Fig. 2). Screening of an
S. pombe genomic library unexpectedly identified two related
genes that encode proteins with homology to WD repeat-containing TAFs
such as hTAF100, dTAF80/85, and yTAF90 (Fig.
3). One gene (represented by five clones)
was found to be taf72+, a putative TAF gene
isolated on the basis of sequence similarity (35). The
taf72+ gene encodes a protein of 643 amino acids
with a predicted molecular mass of 72.4 kDa, but its association with
TBP has not been demonstrated. The other gene (represented by two
clones) was a previously unidentified gene, which has been named
taf73+. A comparison between the genomic DNA and
cDNA sequences of the taf73+ gene revealed
that there is a 56-base pair intron (nucleotides 1474-1529) with
consensus sequences for splicing (41).
The taf73+ gene encodes a protein of 642 amino
acids with a predicted molecular mass of 72.3 kDa. Like other WD
repeat-containing TAFs, TAF73 contains six WD repeats (23) in its
C-terminal half (Fig. 3B). The TAF73 protein is 45%
identical to TAF72, 39% identical to yTAF90, 30% identical to
hTAF100, and 29% identical to dTAF80/85. Similarity was observed
throughout the proteins, although the C-terminal regions show higher
degrees of conservation (Fig. 3B). TAF72 is more similar to
yTAF90, hTAF100, and dTAF80/85 than TAF73 is. We also compared TAF72
and TAF73 with PAF65
To test the possibility that the suppression of the
ubcP4ts mutation by taf72+ or
taf73+ results from an increase in
ubcP4 expression, we carried out Northern analysis.
ubcP4 mRNA levels were not affected by multicopy plasmids carrying the taf72+ or
taf73+ gene (Fig.
4). We speculate that the suppression
results from increased expression of some other genes involved in
APC/C-mediated proteolysis (see "Discussion"). Suppression of the
ubcP4ts mutation seems to be specific to the
taf72+ and taf73+ genes
because no other TAF genes were isolated in our library screen.
TAF72 and TAF73 Have Nonredundant Functions--
The
taf72+ gene is essential for cell viability
(35). To determine whether taf73+ is also an
essential gene, we constructed a diploid S. pombe strain in
which one copy of taf73+ was disrupted. The
taf73+/
We next examined whether overexpression of TAF72 suppresses
TAF72 and TAF73 Are Associated with TBP and Other TAFs--
To
detect TAF72 and TAF73 proteins, we constructed S. pombe
strains expressing FLAG-tagged TAF72 or TAF73, in which the wild-type gene on the chromosome was replaced by a gene encoding the
epitope-tagged protein (see "Experimental Procedures"). Whole-cell
extracts were prepared from these strains, along with wild-type strain
JY741, which did not express any FLAG-tagged protein. Immunoblotting with anti-FLAG antibody detected FLAG-TAF72 and FLAG-TAF73 proteins, which were absent from the extract of the wild-type strain (data not
shown). FLAG-TAF72 and FLAG-TAF73 migrated on SDS-polyacrylamide gel
with apparent molecular masses of ~75 and ~80 kDa, respectively.
We next examined whether TAF72 and TAF73 are associated with TBP. The
whole-cell extracts of the strains expressing FLAG-TAF72 or FLAG-TAF73
were used for immunoprecipitation with anti-FLAG antibody.
Immunoblotting with anti-S. pombe TBP antibody revealed that
TBP was co-immunoprecipitated with TAF72 and TAF73 (Fig. 6A, lanes 2 and
3). TBP was not immunoprecipitated by anti-FLAG antibody
from the extract of the wild-type strain (Fig. 6A,
lane 1), although TBP was present in the extract (data not
shown). We also tested association of TAF72 and TAF73 with two other
TAFs in S. pombe, TAF130 and PTR6. TAF130 is a homolog of
hTAF250, dTAF230/250, and
yTAF130/145,2 and PTR6 is a
putative TAF that has homology to hTAF55 and yTAF67 (42).
Immunoblotting with anti-TAF130 and anti-PTR6 antibodies showed that
TAF130 and PTR6 were also co-immunoprecipitated with TAF72 and TAF73
(Fig. 6A, lanes 2 and 3). These
results indicate that TAF72 and TAF73 are each present in a complex(es)
with TBP, TAF130, and PTR6. Since TAF72 and TAF73 were associated with
TAF130, a homolog of TFIID-specific TAFs, it is most likely that TAF72 and TAF73 are components of the S. pombe TFIID complex.
Conversely, we carried out immunoprecipitation with anti-TBP antibody.
Immunoblotting with anti-FLAG, anti-TAF130, and anti-PTR6 antibodies
revealed that TAF72, TAF73, TAF130, and PTR6 were co-immunoprecipitated with TBP (Fig. 6B, lanes 2 and 4).
These TAFs were not immunoprecipitated when preimmune serum was used
instead of anti-TBP serum (Fig. 6B, lanes 1 and
3). These results demonstrate that TAF72, TAF73, TAF130, and
PTR6 are each associated with TBP and therefore, by definition, are TAFs.
TAF72 and TAF73 Are Present in the Same Complex--
We next asked
whether TAF72 and TAF73 are present in the same complex or in distinct
complexes. To address this question, we constructed a diploid S. pombe strain expressing both FLAG-tagged TAF72 and HA-tagged
TAF73. Immunoprecipitation with anti-FLAG antibody followed by
immunoblotting with anti-HA antibody revealed that HA-TAF73 was
co-immunoprecipitated with FLAG-TAF72 (Fig. 6C, lane
2). HA-TAF73 was not immunoprecipitated from the extract of a
strain expressing HA-TAF73, but not FLAG-TAF72 (Fig. 6C, lane 1). These results clearly indicate that TAF72 and TAF73
are present in the same complex. We then used a diploid strain
expressing both FLAG-tagged TAF73 and HA-tagged TAF73 to test the
possibility that two copies of TAF73 are present in the same
complex. HA-TAF73 was not co-immunoprecipitated with FLAG-TAF73 (Fig.
6C, lane 3), indicating that the complex contains
only one molecule of TAF73.
TAF72, but Not TAF73, Is Associated with Gcn5--
yTAF90, the
S. cerevisiae homolog of TAF72 and TAF73, is also present in
SAGA, a histone acetylase complex distinct from TFIID (24). We tested
the possibility that TAF72 and TAF73 are shared by TFIID and other
histone acetylase complexes. In S. pombe, a SAGA-like
complex has not been characterized. However, data base searches
revealed that the S. pombe genome contains genes that encode
proteins homologous to SAGA subunits. For example, the SPAC1952.05
gene, which has been predicted by the S. pombe Genome Sequencing Project, encodes a 454-amino acid protein that is 53% identical and 69% similar to S. cerevisiae Gcn5 (439 amino
acids), the histone acetylase subunit of the SAGA complex. As shown in Fig. 7A, there is a high
degree of conservation between S. cerevisiae Gcn5 and its
putative S. pombe homolog except for the N-terminal region,
which is dispensable for S. cerevisiae Gcn5 function
in vivo (43). We refer to this gene as
gcn5+ and examined whether its product (Gcn5) is
associated with TAF72 and TAF73.
We replaced the gcn5+ gene of the wild-type,
taf72FLAG, and taf73FLAG
strains with gcn5HA, which encodes HA-tagged
Gcn5 protein, and prepared whole-cell extracts from the strains
expressing HA-Gcn5; HA-Gcn5 and FLAG-TAF72; or HA-Gcn5 and FLAG-TAF73.
Immunoblotting with anti-HA antibody detected HA-Gcn5 protein in the
whole-cell extracts (Fig. 7B, lanes 1-3).
Immunoprecipitation with anti-FLAG antibody followed by immunoblotting
with anti-HA antibody revealed that Gcn5 was co-immunoprecipitated with
TAF72, but not with TAF73 (Fig. 7B, lanes 5 and
6). These results indicate that TAF72 is associated with
Gcn5. Thus, it is likely that S. pombe has a SAGA-like
complex that shares TAF72 with TFIID. Interestingly, in contrast to
TAF72, TAF73 is not present in the putative SAGA-like complex. The
association of Gcn5 with TAF72 is consistent with the prediction that
the gcn5+ gene (SPAC1952.05) encodes a homolog
of S. cerevisiae Gcn5.
Overexpression of TAF72 or TAF73 Suppresses a cut9ts
Mutation--
The ubiquitin-conjugating enzyme UbcP4 seems to be
involved in APC/C-mediated proteolysis during mitosis because depletion (29) or inactivation (see above) of UbcP4 results in an anaphase block
similar to those caused by mutations in the APC/C subunit genes such as
cut9-665 and because overexpression of UbcP4 suppresses the
cut9-665 mutation (29). We asked whether
taf72+ and taf73+ are
able to suppress the cut9-665 mutation as well as the
ubcP4-140 mutation. As shown in Fig.
8A, multicopy plasmids
carrying the taf72+ or
taf73+ gene suppressed the temperature-sensitive
growth of a cut9-665 mutant. This did not seem to result
from an increase in ubcP4 expression because the
taf72+ and taf73+
plasmids did not affect ubcP4 mRNA levels (Fig. 4). We
tested analogous suppression in S. cerevisiae using the
TAF90 gene (the taf72+ and
taf73+ homolog) and a mutation in the
CDC16 gene (the cut9+ homolog). A
multicopy plasmid carrying the TAF90 gene did not suppress
the temperature-sensitive growth of a cdc16-1 mutant at
30 °C on synthetic or complex medium (data not shown).
As described above, taf72+ and
taf73+ are able to suppress mutations in both a
ubiquitin-conjugating enzyme gene (ubcP4+) and a
ubiquitin-protein ligase complex subunit gene
(cut9+). To test specificity of suppression, we
examined whether taf72+ and
taf73+ suppress a mutation in another gene
involved in ubiquitin-dependent proteolysis. The
mts2+ gene encodes a homolog of the human S4
subunit of the 19 S complex of the proteasome (44), and the
temperature-sensitive mts2-1 mutation causes defects in
chromosome segregation and in ubiquitin-dependent proteolysis (31). Neither the taf72+ nor
taf73+ plasmid suppressed the mts2-1
mutation on EMM or YE + AdeUra medium (Fig. 8B and data not shown).
In this study, we have identified two WD repeat-containing TAFs in
fission yeast that may regulate genes involved in cell cycle progression.
TAF in Fission Yeast--
In S. pombe, two putative TAF
genes, taf72+ and ptr6+,
have been reported thus far. taf72+ was cloned
by PCR on the basis of homology to WD repeat-containing TAFs (35); and
ptr6+ was identified through a genetic screen
for mutants defective in poly(A)+ RNA transport (42).
However, neither TAF72 nor PTR6 has been shown to be associated with
TBP. T. Kokubo and colleagues have identified another TAF
gene, taf130+, which encodes the S. pombe homolog of hTAF250, dTAF230/250, and
yTAF130/145.2 In this study, we have isolated and
characterized taf72+ and a new gene named
taf73+. We demonstrated that TAF72, TAF73,
TAF130, and PTR6 are all associated with TBP. Thus, we conclude that
these proteins are indeed TAFs. To our knowledge, this is the first
report of biochemical characterization of TAFs in S. pombe.
Data base searches revealed that the S. pombe genome
contains many putative TAF genes, including those encoding homologs of
human TAF150, TAF70/80, TAF31/32, TAF30, TAF28, TAF20, and TAF18 (data
not shown). It is thus most likely that S. pombe has a TFIID
complex(es) similar to those identified in human,
Drosophila, and S. cerevisiae. The association of
TAF72 and TAF73 with TAF130, a homolog of TFIID-specific TAFs, suggests that TAF72 and TAF73 are components of the S. pombe TFIID
complex. It has been shown that there are multiple forms of TFIID
complexes (45). We showed that TFIID in S. pombe
contains both TAF72 and TAF73. It remains to be determined, however,
whether S. pombe has multiple TFIID complexes.
Our results have implications for the stoichiometry of WD
repeat-containing TAFs in the TFIID complex. We showed that TAF72 and
TAF73 are present in the same complex. Unlike S. pombe
TFIID, the human, Drosophila, and S. cerevisiae
TFIID complexes contain a single species of WD repeat-containing TAF:
hTAF100, dTAF80/85, and yTAF90, respectively. We speculate that these
TAFs might be present in two copies in TFIID.
Subsets of TAFs have been found in histone acetylase complexes distinct
from TFIID (3). WD repeat-containing TAFs are present in non-TFIID
complexes such as SAGA and TFTC (24, 25). We showed that TAF72 is
associated with Gcn5, a homolog of the histone acetylase subunit of the
S. cerevisiae SAGA complex. In contrast to TAF72, TAF73 is
not associated with Gcn5. It seems that TAF72 is present in both TFIID
and SAGA-like complexes, whereas TAF73 is present only in TFIID.
TAF and Cell Cycle--
The taf72+ and
taf73+ genes were isolated as multicopy
suppressors of a mutation in the ubiquitin-conjugating enzyme gene
ubcP4+. taf72+ and
taf73+ also suppressed a mutation in the
ubiquitin-protein ligase complex subunit gene
cut9+. Overexpression of TAF72 or TAF73 from the
expression vector pREP1 carrying the taf72+ or
taf73+ cDNA inhibited cell growth
(taf73+ was more inhibitory than
taf72+) (data not shown). We think that this is
due to interference of TFIID function. In contrast, suppression was
observed for multicopy plasmids carrying the genomic DNA fragment of
taf72+ or taf73+, and
these plasmids did not inhibit cell growth (probably because of lower
expression levels). Thus, it is likely that moderate overexpression
leads to suppression, but higher expression is deleterious to the cell.
We infer that moderate overexpression of TAF72 or TAF73 might lead to
an elevated level of TFIID or other TAF-containing complexes, which in
turn results in increased expression of certain genes involved in
APC/C-mediated proteolysis. Transcription might be generally increased
upon overexpression of TAF72 or TAF73. Alternatively, overexpression of
TAF72 or TAF73 might affect the transcription of only a subset of
genes. We favor a model in which TAF72 and TAF73 are specifically
required for the expression of a subset of genes involved in cell cycle
progression through mitosis, including those involved in APC/C-mediated
proteolysis, because yTAF90, the S. cerevisiae homolog of
TAF72 and TAF73, is not generally required for transcription (15, 46),
and a taf90ts mutation causes cell cycle
arrest at G2/M (7).
Genome-wide expression analysis was carried out with the
taf90ts mutation to identify genes whose
expression depends on yTAF90 (15). The yTAF90-dependent
genes include APC2, an APC/C subunit gene, which might
explain the G2/M arrest phenotype caused by the
taf90ts mutation. Since yTAF90 is shared by
TFIID and SAGA, the transcription defects caused by yTAF90 inactivation
should reflect yTAF90 function in both TFIID and SAGA. Unlike yTAF90,
TAF73 is not present in SAGA. Therefore, TAF73 will provide a good
model for understanding the in vivo function of WD
repeat-containing TAFs in TFIID.
As discussed above, TAF72 and TAF73 may, directly or indirectly,
regulate the expression of genes involved in APC/C-mediated proteolysis. Northern analysis indicated that ubcP4 mRNA
levels did not increase upon overexpression of TAF72 or TAF73.
cut9+ or other APC/C subunit genes may be
regulated. A multicopy suppressor of the cut9-665 mutation,
hcn1+, has been reported that encodes a protein
homologous to the S. cerevisiae APC/C subunit Cdc26 (47).
Since Cut9 function is regulated by the protein kinase A pathway (47),
possible candidates include genes involved in this pathway. It should
be noted that the expression of TAF72- or TAF73-dependent
genes is not necessarily cell cycle-regulated. In fact, genome-wide
expression analyses in S. cerevisiae showed that the
expression of most of the genes involved in APC/C-mediated proteolysis
is not cell cycle-regulated, although some (for example,
APC1 and CDC20) show cell cycle fluctuation (48,
49). In contrast, the expression of many APC/C subunit genes
(APC4, APC5, APC9, APC11,
CDC16, CDC23, CDC26, and
CDC27) and APC/C activator genes (CDC20 and
CDH1/HCT1) is induced through sporulation (meiosis)
(50).
Our results provide evidence for a possible role of WD
repeat-containing TAFs in the expression of genes involved in
progression through the M phase of the cell cycle. Genes that require
TAF72 or TAF73 function remain to be identified. Conditional lethal mutations would be useful to analyze the cell cycle phenotype and gene
expression upon inactivation of TAF72 or TAF73.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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, is present in the
p300/CBP-associated factor (PCAF) and TFTC complexes (25, 26).
EXPERIMENTAL PROCEDURES
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ade6-M216 leu1 ura4-D18), JY746
(h+ ade6-M210 leu1 ura4-D18),
ubcP4ts (h
ade6-M210 ura4 leu1
ubcP4-140::ura4+) (this study),
cut9ts (h
leu1 cut9-665; a
gift from Mitsuhiro Yanagida) (30), and mts2ts
(h+ leu1-32 mts2-1; a gift from Colin
Gordon) (31). S. pombe media were prepared as described (32,
33). Standard methods were used for molecular genetic analysis of
S. pombe (32, 33).
SS, a pUC19 derivative
lacking the SspI site. A ura4+
fragment was inserted into the SspI site located 440 base
pairs downstream of the ubcP4+ gene. A
ubcP4+::ura4+
fragment was amplified by PCR in the presence of 0.5 mM
MnCl2 and used to transform an S. pombe ura4
strain. Ura+ transformants selected at 25 °C were
replica-plated onto EMM + AdeLeu containing phloxine B (Sigma), and
the plates were incubated at 36 °C. Inability of one clone to grow
at 36 °C was complemented by the pREP81-ubcP4 plasmid, indicating
that the clone contains a recessive, temperature-sensitive mutation in
the ubcP4+ gene, which was designated
ubcP4-140. Replacement of ubcP4+ by
ubcP4-140::ura4+ was
confirmed by Southern analysis. Sequencing of the ubcP4-140 allele identified two amino acid substitutions: isoleucine by threonine
at position 80 and threonine by alanine at position 129.
taf73::ura4). A 2.7-kb blunt-end
taf73::ura4+ fragment
was amplified from pGEM-T(
taf73::ura4) by PCR with Vent
DNA polymerase (New England Biolabs) and used for the one-step gene
disruption (36). After transformation, Ura+ colonies were
screened for sensitivity to 5-fluoroorotic acid (Toronto Research
Chemicals). Correct disruption was confirmed by PCR. Complementation by
a taf73+ plasmid (Table I) also confirmed
correct disruption.
80 °C. The cells
(~350 mg) were resuspended in 700 µl of buffer A (20 mM
HEPES-KOH (pH 7.6), 150 mM potassium acetate, 20%
glycerol, 0.1% Nonidet P-40, and 1 mM dithiothreitol) with
1 mM phenylmethylsulfonyl fluoride (or
4-(2-aminoethyl)benzenesulfonyl fluoride) and protease inhibitor
mixture (Roche Molecular Biochemicals) and then disrupted with 1 ml of
glass beads (~0.5 mm in diameter) six times for 1 min using a
BeadBeater (BioSpec Products). Cell lysates were recovered through a
small hole punched at the bottom of the tube and clarified twice by
centrifugation at 18,000 × g for 10 min. The protein concentration of the extracts was typically ~20 mg/ml.
80 °C. Proteins that had derived
from 750 or 1500 µg of total protein were separated by 7.5 or 10%
SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene
fluoride membrane, and probed with antibodies. Immune complexes were
detected by chemiluminescence. In some cases, a blot was stripped
before reprobing.
RESULTS
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DISCUSSION
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Fig. 1.
A temperature-sensitive ubcP4
mutant. A, growth of wild-type ( ) and
ubcP4-140 (
) strains at the nonpermissive temperature.
Log-phase cultures in YE medium were shifted from 25 to 36 °C. Cells
were counted with a hemocytometer. B, uncoordinated mitosis
of the ubcP4ts mutant. Cells were fixed with
methanol at 4 h (panel b) or 6 h (panel
a) after the shift to 36 °C and stained with
4',6-diamidino-2-phenylindole. The arrow indicates a
metaphase-arrested cell with condensed chromosomes. The closed
arrowhead indicates a septated cell without chromosome
segregation, and the open arrowhead indicates a cell
undergoing cytokinesis without chromosome segregation (a cut
phenotype). Bar = 10 µm.
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Fig. 2.
Suppression of a ubcP4ts
mutation by multicopy plasmids carrying the
taf72+ or
taf73+ gene. A ubcP4-140
strain was transformed with pSP1 (vector), pSP1 carrying the
taf72+ gene, pSP1 carrying the
taf73+ gene, or pSP1 carrying the
ubcP4+ gene, and the transformants were grown on
EMM + Ade for 4 days at 25 °C (left) and 35 °C
(right). The taf72+ and
taf73+ plasmids contain the subcloned fragments
shown in Fig. 3A.
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Fig. 3.
Multicopy suppressors of
ubcP4ts that encode WD repeat-containing
TAFs. A, genomic DNA fragments capable of suppressing
the ubcP4ts mutation. Subcloning of inserts from two
plasmids (pSP1-19 and pSP1-7) that suppressed the ubcP4-140
mutation resulted in the 2.2-kb SalI-HindIII
fragment containing the taf73+ gene and the
2.6-kb SacI-HindIII fragment containing the
taf72+ gene. Exons are indicated by black
boxes. B, amino acid sequence alignment of WD
repeat-containing TAFs and a related PAF. Sequences are from S. pombe (sp) TAF73 (DDBJ/EMBL/GenBankTM Data
Bank accession number AB039954) and TAF72 (accession number AB001372),
yTAF90 (accession number Z36067), hTAF100 (accession number U80191),
PAF65 (accession number AF069736), and dTAF80 (accession number
U06460). The alignment was generated with the ClustalW program.
Identical and similar residues were shaded with the program
Boxshade. The arrows below the sequences indicate WD
repeats.
, an hTAF100-related protein present in the
human histone acetylase complexes PCAF and TFTC (25, 26). Both TAF72
and TAF73 are less related to PAF65
(27 and 22% identical,
respectively) than to hTAF100 (34 and 30% identical).
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Fig. 4.
No effect of TAF72 or TAF73 overexpression on
ubcP4 transcription. Total RNA was isolated from
a wild-type strain transformed with pSP1 (vector), pSP1-7
(taf72+), pSP1-19
(taf73+), or pSP1 carrying the
ubcP4+ gene and was analyzed by Northern
blotting using 32P-labeled ubcP4+
and cdc2+ cDNAs as probes.
taf73::ura4+
cells were sporulated and subjected to tetrad analysis. Of 34 tetrads
dissected, 0, 1, and 2 viable spores were observed for 2, 15, and 17 tetrads, respectively, and no tetrads with more than 2 viable spores
were recovered (Fig. 5). Importantly, all the viable spores were Ura
and thus presumed to be
taf73+. Microscopic observation of the 34
taf73::ura4+ spores
revealed that most spores germinated and divided three times before
they ceased growing (no spores divided more than four times). In
addition,
taf73::ura4+
haploid cells carrying a taf73 plasmid,
pREP81(taf73cDNA), did not lose the plasmid under nonselective
conditions. These results indicate that the
taf73+ gene, like taf72+,
is essential for cell viability. The
taf73 strain
carrying the plasmid pREP81(taf73cDNA) grew even under conditions
that repress taf73+ expression (i.e.
in the presence of thiamine), indicating that residual expression
allows cells to grow. Consequently, whether depletion of TAF73 causes a
cell cycle phenotype remains to be determined.
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Fig. 5.
Disruption of the
taf73+ gene. The
taf73::ura4+ allele was
created by replacing the entire coding region of
taf73+ with a ura4+
fragment as described under "Experimental Procedures." The
taf73+/
taf73::ura4+
diploid cells were sporulated on malt extract medium at 27 °C
for 2 days. Tetrads were dissected on YE + AdeUra medium, and spores
were grown at 30 °C for 3 days. Ten tetrads are shown; the four
spores from each tetrad are aligned vertically.
taf73. The
taf73+/
taf73::ura4+
diploid strain was transformed with multicopy plasmids carrying the
taf72+ or taf73+ gene,
sporulated, and subjected to tetrad analysis. As shown in Table
I, tetrads with more than two viable
spores were recovered from the diploid carrying the
taf73+ plasmid, but not from the diploid
carrying the taf72+ plasmid, indicating that
overexpression of TAF72 did not suppress
taf73. Thus,
TAF72 cannot substitute for TAF73.
Overexpression of TAF72 does not suppress taf73
taf73::ura4+
diploid was transformed with multicopy plasmids carrying the
taf72+ or taf73+ gene.
Transformants with pSP1 (Vector), pSP1-7 (taf72+),
or pSP1-19 (taf73+) were sporulated and subjected
to tetrad analysis.
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Fig. 6.
Association of TAF72 and TAF73 with TBP.
Immunoprecipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis, transferred, and probed as described under
"Experimental Procedures." A, co-immunoprecipitation of
TBP with TAF72 or TAF73. Whole-cell extracts prepared from wild-type
strain JY741 (WT; lane 1) and strains expressing
FLAG-TAF73 (taf73FLAG; lane 2) or
FLAG-TAF72 (taf72FLAG; lane 3) were
used for immunoprecipitation (IP) with antibody to FLAG,
followed by immunoblotting with antibodies to FLAG, TBP, TAF130, and
PTR6. B, co-immunoprecipitation of TAF72 or TAF73 with TBP.
Whole-cell extracts prepared from strains expressing FLAG-TAF73
(taf73FLAG; lanes 1 and 2)
or FLAG-TAF72 (taf72FLAG; lanes 3 and
4) were used for immunoprecipitation with preimmune serum
(PI; lanes 1 and 3) or anti-TBP
antibody (TBP; lanes 2 and 4),
followed by immunoblotting with antibodies to TBP, FLAG, TAF130, and
PTR6. C, co-immunoprecipitation of TAF73 with TAF72.
Whole-cell extracts prepared from diploid strains expressing HA-TAF73
(taf73HA × WT; lane 1),
HA-TAF73 and FLAG-TAF72 (taf73HA × taf72FLAG; lane 2), or HA-TAF73 and
FLAG-TAF73 (taf73HA × taf73FLAG; lane 3) were used for
immunoprecipitation with antibody to FLAG, followed by immunoblotting
with antibodies to FLAG and HA.
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Fig. 7.
Association of TAF72 with Gcn5.
A, amino acid sequence alignment of the S. cerevisiae histone acetylase Gcn5 (Sc) and its putative
homolog in S. pombe (Sp). Sp, the
product encoded by the predicted gene SPAC1952.05
(DDBJ/EMBL/GenBankTM Data Bank accession number AL109820);
Sc, Gcn5/YGR252W (accession number Z73037). B,
co-immunoprecipitation of Gcn5 with TAF72, but not with TAF73.
Whole-cell extracts (WCE) prepared from strains expressing
HA-Gcn5 (gcn5HA; lanes 1 and
4), HA-Gcn5 and FLAG-TAF72 (gcn5HA
taf72FLAG; lanes 2 and 5),
or HA-Gcn5 and FLAG-TAF73 (gcn5HA
taf73FLAG; lanes 3 and 6)
were used for immunoprecipitation (IP) with antibody to
FLAG, followed by immunoblotting with antibodies to FLAG and HA.
Lanes 1-3, whole-cell extracts (2.5% of input);
lanes 4-6, immunoprecipitated proteins.
Arrowheads indicate the positions of FLAG-TAF72
(lower) and FLAG-TAF73 (upper).
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Fig. 8.
Specific suppression of a
cut9ts mutation by overexpression of TAF72 or
TAF73. A, suppression of a cut9ts
mutation by multicopy plasmids carrying the
taf72+ or taf73+ gene. A
cut9-665 strain was transformed with pSP1
(vector), pSP1-7 (taf72+), or pSP1-19
(taf73+), and the transformants were grown on
EMM at 31 °C for 2 days. Four transformants for each plasmid are
shown. Transformants with the taf72+ plasmid
grew better than those with the taf73+ plasmid.
Similar suppression was observed on YE + AdeUra medium at 31 °C for
the taf72+ plasmid, but not for the
taf73+ plasmid (data not shown). B,
no suppression of an mts2ts mutation by multicopy
plasmids carrying the taf72+ or
taf73+ gene. An mts2-1 strain was
transformed with pSP1 (vector), pSP1-7
(taf72+), or pSP1-19
(taf73+), and the transformants were grown on
EMM at 31 °C for 2 days. Two transformants for each plasmid are
shown.
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ACKNOWLEDGEMENTS |
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We thank Tetsuro Kokubo for antibodies to TBP, TAF130, and PTR6 and for a TAF90 plasmid; Mitsuhiro Yanagida for a cut9ts strain; Colin Gordon for an mts2ts strain; and Tokio Tani for a ptr6+ plasmid. We also thank Yoshinori Watanabe and Masayuki Yamamoto for S. pombe strains and plasmids. We are grateful to Phil Hieter, Kim Nasmyth, and Akio Toh-e for S. cerevisiae cdc strains. We are also indebted to Hisako Suzuki for help with DNA sequencing and to Nicole Robinson for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Science and Technology Corp.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 nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank under accession number AB039954.
§ To whom correspondence should be addressed. Tel.: 81-559-81-6744; Fax: 81-559-81-6746; E-mail: hmitsuza@lab.nig.ac.jp.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100248200
2 T. Kokubo, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: TF, transcription factor; TBP, TATA-binding protein; TAF, TBP-associated factor (prefixes y, h, and d indicate yeast (S. cerevisiae), human, and Drosophila, respectively); SAGA, Spt-Ada-Gcn5 acetyltransferase; TFTC, TBP-free TAFII-containing; PCAF, p300/CBP-associated factor; PAF, PCAF-associated factor; APC/C, anaphase-promoting complex or cyclosome; PCR, polymerase chain reaction; EMM, Edinburgh minimal medium; kb, kilobase pair(s); HA, hemagglutinin; YE, yeast extract; PTR, poly(A)+ RNA transport.
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