Two WD repeat-containing TATA-binding protein-associated factors in fission yeast that suppress defects in the anaphase-promoting complex.

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 ubcP4(ts) 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).
Ubiquitin-dependent proteolysis has been shown to play a key role in progression through the cell cycle (27). A ubiquitinprotein 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 ϩ , 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 ϩ and taf73 ϩ , 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 ϩ and taf73 ϩ suppress a mutation in the cut9 ϩ 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.
Isolation of a ubcP4 ts Mutation-An XhoI-XbaI fragment containing the ubcP4 ϩ gene was cloned into the SalI/XbaI site of pUC19⌬SS, a pUC19 derivative lacking the SspI site. A ura4 ϩ fragment was inserted into the SspI site located 440 base pairs downstream of the ubcP4 ϩ gene. A ubcP4 ϩ ::ura4 ϩ fragment was amplified by PCR in the presence of 0.5 mM MnCl 2 and used to transform an S. pombe ura4 strain. Ura ϩ transformants selected at 25°C were replica-plated onto EMM ϩ Ade-Leu 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 ϩ gene, which was desig-nated ubcP4-140. Replacement of ubcP4 ϩ by ubcP4-140::ura4 ϩ 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.
Isolation and Characterization of Multicopy Suppressors of a ubcP4 ts Mutation-A ubcP4-140 strain was transformed with an S. pombe genomic library constructed with the multicopy plasmid pSP1 (34). Leu ϩ transformants selected on EMM ϩ Ade at 25°C were replicaplated onto EMM ϩ 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 ϩ gene (35) and the latter contained a related but previously unidentified gene. This gene was named taf73 ϩ and analyzed further. taf73 ϩ cDNA was amplified by reverse transcription-PCR, cloned into pGEM-T (Promega), and sequenced. A 3Ј-portion of taf73 ϩ cDNA was also amplified by 3Ј-rapid amplification of cDNA ends and sequenced.
Disruption of the taf73 ϩ Gene-A 3.0-kb genomic DNA fragment containing the taf73 ϩ gene was amplified by PCR and cloned into pGEM-T to generate pGEM-T(taf73). The entire vector sequence flanked by 5Ј-and 3Ј-noncoding sequences of taf73 ϩ was amplified from pGEM-T(taf73) by PCR, digested with XhoI and SmaI, and ligated with a 1.8-kb XhoI-SmaI ura4 ϩ fragment to generate pGEM-T(⌬taf73::ura4). A 2.7-kb blunt-end ⌬taf73::ura4 ϩ 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 ϩ colonies were screened for sensitivity to 5-fluoroorotic acid (Toronto Research Chemicals). Correct disruption was confirmed by PCR. Complementation by a taf73 ϩ plasmid (Table I) also confirmed correct disruption.
Construction of S. pombe Strains Expressing Epitope-tagged TAF or Gcn5-To construct S. pombe strains expressing FLAG or HA epitopetagged 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 5Ј-noncoding region and a 5Ј-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 ϩ fragment. The resulting plasmids were linearized at a unique restriction site within the taf genes (AatII for taf72 ϩ and SpeI for taf73 ϩ ) (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 3Ј-truncated taf gene that are separated by the ura4 ϩ 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 taf FLAG strains. Similarly, an S. pombe strain ex-  pressing 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 taf73 HA strain was crossed with the wild-type, taf72 FLAG , and taf73 FLAG 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, FIG. 3. Multicopy suppressors of ubcP4 ts that encode WD repeat-containing TAFs. A, genomic DNA fragments capable of suppressing the ubcP4 ts 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 ϩ gene and the 2.6-kb SacI-HindIII fragment containing the taf72 ϩ 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/GenBank TM 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.
were constructed using the wild-type, taf72 FLAG , and taf73 FLAG strains. A DNA fragment containing a 5Ј-noncoding region and a 5Ј-portion of the coding sequence with the triple HA sequence immediately after the initiation codon was cloned into the ura4 ϩ 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 A 600 of 0.5 (1 ϫ 10 7 cells/ml) in 200 ml of YE ϩ AdeUra medium at 30°C. Cells were harvested, washed with H 2 O, transferred to a 2.0-ml tube, and frozen at Ϫ80°C. The cells (ϳ350 mg) were resuspended in 700 l 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 (ϳ0.5 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 ϫ g for 10 min. The protein concentration of the extracts was typically ϳ20 mg/ml.
Immunoprecipitation and Immunoblotting-For immunoprecipitation with anti-FLAG antibody, 300 -500 l 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 l of whole-cell extract was mixed with 10 l of anti-S. pombe TBP serum or preimmune serum and incubated for 1 h on ice, and then 40 l 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 1.5ϫ SDS gel loading buffer, and frozen at Ϫ80°C. Proteins that had derived from 750 or 1500 g 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.
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 antirabbit 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 ϩ gene has been submitted to the DDBJ/EMBL/GenBank TM 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 ϩ gene was determined by the S. pombe Genome Sequencing Project. Our taf73 ϩ sequence is identical to the sequence of the predicted gene SPBC15D4.14 on cosmid c15D4 (accession number AL031349).

RESULTS
Isolation of Putative TAF Genes as Multicopy Suppressors of a ubcP4 ts 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 ϩ gene (designated ubcP4-140 or ubcP4 ts ) 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, ϳ30% of the cells showed septation or cytokinesis without chromosome segregation. Thus, the ubcP4 ts 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 ϩ , a putative TAF gene isolated on the basis of sequence similarity (35). The taf72 ϩ 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 ϩ . A comparison between the genomic DNA and cDNA sequences of the taf73 ϩ gene revealed that there is a 56-base pair intron (nucleotides 1474 -1529) with consensus sequences for splicing (41).
FIG. 5. Disruption of the taf73 ؉ gene. The ⌬taf73::ura4 ϩ allele was created by replacing the entire coding region of taf73 ϩ with a ura4 ϩ fragment as described under "Experimental Procedures." The taf73 ϩ /⌬taf73::ura4 ϩ diploid cells were sporulated on malt extract medium at 27°C for 2 days. Tetrads were dissected on YE ϩ 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. than to hTAF100 (34 and 30% identical).
To test the possibility that the suppression of the ubcP4 ts mutation by taf72 ϩ or taf73 ϩ results from an increase in ubcP4 expression, we carried out Northern analysis. ubcP4 mRNA levels were not affected by multicopy plasmids carrying the taf72 ϩ or taf73 ϩ 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 ubcP4 ts mutation seems to be specific to the taf72 ϩ and taf73 ϩ genes because no other TAF genes were isolated in our library screen.
TAF72 and TAF73 Have Nonredundant Functions-The taf72 ϩ gene is essential for cell viability (35). To determine whether taf73 ϩ is also an essential gene, we constructed a diploid S. pombe strain in which one copy of taf73 ϩ was disrupted. The taf73 ϩ /⌬taf73::ura4 ϩ 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 Ϫ and thus presumed to be taf73 ϩ . Microscopic observation of the 34 ⌬taf73::ura4 ϩ 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 ϩ haploid cells carrying a taf73 plasmid, pREP81(taf73cDNA), did not lose the plasmid under nonselective conditions. These re-sults indicate that the taf73 ϩ gene, like taf72 ϩ , is essential for cell viability. The ⌬taf73 strain carrying the plasmid pREP81(taf73cDNA) grew even under conditions that repress taf73 ϩ 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.
We next examined whether overexpression of TAF72 suppresses ⌬taf73. The taf73 ϩ /⌬taf73::ura4 ϩ diploid strain was transformed with multicopy plasmids carrying the taf72 ϩ or taf73 ϩ 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 ϩ plasmid, but not from the diploid carrying the taf72 ϩ plasmid, indicating that overexpression of TAF72 did not suppress ⌬taf73. Thus, TAF72 cannot substitute for TAF73.
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  a Ura ϩ spores are presumed to be ⌬taf73ϻura4 ϩ segregants. All viable Ura ϩ spores were also Leu ϩ . b Leu ϩ spores are segregants carrying the plasmid. c For unknown reasons, the taf72 ϩ plasmid increased the spore viability of taf73 ϩ segregants.
strain (data not shown). FLAG-TAF72 and FLAG-TAF73 migrated on SDS-polyacrylamide gel with apparent molecular masses of ϳ75 and ϳ80 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 TFIIDspecific TAFs, it is most likely that TAF72 and TAF73 are components of the S. pombe TFIID complex.
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 FLAGtagged 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 ϩ and examined whether its product (Gcn5) is associated with TAF72 and TAF73.
We replaced the gcn5 ϩ gene of the wild-type, taf72 FLAG , and taf73 FLAG strains with gcn5 HA , 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 2 T. Kokubo, personal communication.
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 ϩ gene (SPAC1952.05) encodes a homolog of S. cerevisiae Gcn5.
Overexpression of TAF72 or TAF73 Suppresses a cut9 ts 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 ϩ and taf73 ϩ 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 ϩ or taf73 ϩ 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 ϩ and taf73 ϩ plasmids did not affect ubcP4 mRNA levels (Fig. 4). We tested analogous suppression in S. cerevisiae using the TAF90 gene (the taf72 ϩ and taf73 ϩ homolog) and a mutation in the CDC16 gene (the cut9 ϩ 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 ϩ and taf73 ϩ are able to suppress mutations in both a ubiquitin-conjugating enzyme gene (ubcP4 ϩ ) and a ubiquitin-protein ligase complex subunit gene (cut9 ϩ ). To test specificity of suppression, we examined whether taf72 ϩ and taf73 ϩ suppress a mutation in another gene involved in ubiquitin-dependent proteolysis. The mts2 ϩ 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 ϩ nor taf73 ϩ plasmid suppressed the mts2-1 mutation on EMM or YE ϩ AdeUra medium ( Fig. 8B and data not shown). DISCUSSION 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 ϩ and ptr6 ϩ , have been reported thus far. taf72 ϩ was cloned by PCR on the basis of homology to WD repeat-containing TAFs (35); and ptr6 ϩ was identified through a genetic screen for mutants defective in poly(A) ϩ RNA transport (42). However, neither TAF72 nor PTR6 has been shown to be as-sociated with TBP. T. Kokubo and colleagues have identified another TAF gene, taf130 ϩ , which encodes the S. pombe homolog of hTAF250, dTAF230/250, and yTAF130/145. 2 In this study, we have isolated and characterized taf72 ϩ and a new gene named taf73 ϩ . 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 ϩ and taf73 ϩ genes were isolated as multicopy suppressors of a mutation in the ubiquitin-conjugating enzyme gene ubcP4 ϩ . taf72 ϩ and taf73 ϩ also suppressed a mutation in the ubiquitin-protein ligase complex subunit gene cut9 ϩ . Overexpression of TAF72 or TAF73 from the expression vector pREP1 carrying the taf72 ϩ or taf73 ϩ cDNA inhibited cell growth (taf73 ϩ was more inhibitory than taf72 ϩ ) (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 FIG. 8. Specific suppression of a cut9 ts mutation by overexpression of TAF72 or TAF73. A, suppression of a cut9 ts mutation by multicopy plasmids carrying the taf72 ϩ or taf73 ϩ gene. A cut9-665 strain was transformed with pSP1 (vector), pSP1-7 (taf72 ϩ ), or pSP1-19 (taf73 ϩ ), and the transformants were grown on EMM at 31°C for 2 days. Four transformants for each plasmid are shown. Transformants with the taf72 ϩ plasmid grew better than those with the taf73 ϩ plasmid. Similar suppression was observed on YE ϩ AdeUra medium at 31°C for the taf72 ϩ plasmid, but not for the taf73 ϩ plasmid (data not shown). B, no suppression of an mts2 ts mutation by multicopy plasmids carrying the taf72 ϩ or taf73 ϩ gene. An mts2-1 strain was transformed with pSP1 (vector), pSP1-7 (taf72 ϩ ), or pSP1-19 (taf73 ϩ ), and the transformants were grown on EMM at 31°C for 2 days. Two transformants for each plasmid are shown. taf72 ϩ or taf73 ϩ , 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 taf90 ts mutation causes cell cycle arrest at G 2 /M (7).
Genome-wide expression analysis was carried out with the taf90 ts 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 G 2 /M arrest phenotype caused by the taf90 ts 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/Cmediated proteolysis. Northern analysis indicated that ubcP4 mRNA levels did not increase upon overexpression of TAF72 or TAF73. cut9 ϩ or other APC/C subunit genes may be regulated. A multicopy suppressor of the cut9-665 mutation, hcn1 ϩ , 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 repeatcontaining 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.