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Previous Article | Next Article 
Molecular and Cellular Biology, August 2004, p. 7249-7259, Vol. 24, No. 16
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.16.7249-7259.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology,1 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-23632
Received 13 February 2004/ Returned for modification 16 March 2004/ Accepted 18 May 2004
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ABSTRACT |
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deletion strain has reduced amounts of Ubp8p, and a ubp8 deletion strain shows changes in transcription similar to those seen with the sgf11 deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the four known HAT complexes in yeast, SAGA is the best characterized. The name SAGA refers to its composition of Spt proteins (Spt3p, Spt7p, Spt8p, and Spt20p), Ada proteins (Ada1p, Ada2p, and Ada3p), and Gcn5p acetyltransferase (15). In addition to these components, SAGA also contains Tra1p and a subset of TATA-binding protein associated factors (TAFs), proteins originally identified as members of the TFIID transcription complex (13, 32). These include Taf5p, Taf6p, Taf9p, Taf10p, and Taf12p. A variant of the SAGA complex, named SALSA (SAGA altered, Spt8p absent) or SLIK (SAGA-like), has also been described (35, 42). This version of SAGA lacks Spt8p and has a truncated form of Spt7p. It has been shown that truncation of Spt7p results in loss of Spt8p from the SAGA complex (48). The functional role of SALSA/SLIK is unclear.
The function of SAGA components have been revealed through the study of SAGA-subunit-null strains. DNA microarray experiments suggest that SAGA is required for the expression of 10% of the predicted genes in S. cerevisiae (27). Furthermore, in vivo transcription experiments have pinpointed the importance of SAGA in the regulation of a specific subset of genes including GAL1 (3, 26), HIS3 (2, 42), TRP3 (2, 42), ARG1 (18, 36), and ADH1 (5, 16). Gcn5p is the HAT catalytic subunit of SAGA (12). Gcn5p alone is capable of acetylating free histones; however, acetylation of histone H3 in intact nucleosomes requires SAGA (12). Ada2p and Ada3p regulate SAGA-dependent HAT activity (1, 4, 44). Spt7p, Spt20p, and Ada1p are required for the integrity of SAGA (44, 48). Spt3p and Spt8p mediate the binding of TATA-binding protein to specific promoter regions in vitro and the activation of specific RNA polymerase II-dependent genes in vivo (2, 3). Tra1p is an essential gene and its human homolog TRRAP is a transcription regulatory protein (14, 40).
In a previous study, we described an extensive proteomic investigation of the TFIID complex in S. cerevisiae (41). Individual components of TFIID were immunoaffinity purified from cell extracts, and copurifying proteins were identified by using a high-sensitivity mass spectrometry approach termed direct analysis of large protein complexes (DALPC) (29). Affinity purification of TFIID subunits that are shared with SAGA (Taf5p, Taf6p, Taf9p, Taf10p, and Taf12p) resulted in copurification of components unique to SAGA. In addition to the previously characterized subunits of SAGA, we identified three additional proteins. These include two uncharacterized proteins corresponding to the open reading frames (ORFs) YCL010C and YGL066W (named Sgf29p and Sgf73p, respectively) and a ubiquitin-specific protease, Ubp8p. All three proteins were confirmed as novel SAGA subunits (41).
In the present study, we used a hierarchical clustering algorithm and display developed for identifying gene expression patterns from DNA microarray experiments to reanalyze our proteomic data of proteins copurifying with TFIID (8, 10). The clustering approach arranged proteins according to similarities in patterns of enrichment with the TFIID subunits. Hierarchical clustering provided an unbiased, statistical approach to identifying distinct protein complexes from a large number of proteins that were systematically immunoaffinity purified and analyzed by mass spectrometry. Based on our analysis, an uncharacterized protein corresponding to the ORF YPL047W significantly associates with shared components of TFIID and SAGA. For the present study we have focused on YPL047W (named here as SGF11, 11-kDa SAGA-associated factor) as a novel SAGA component and examined its role in SAGA structure and function. By using mass spectrometry and biochemical assays, we show that Sgf11p interacts with SAGA. We show that Sgf11p is required for the stable association of Ubp8p in purified SAGA. Sgf11p does not appear to regulate the HAT activity of SAGA. However, DNA microarray analysis shows that Sgf11p does mediate transcription of a subset of SAGA-regulated genes. Together, these results indicate that Sgf11p is a novel component of SAGA that mediates gene-specific transcription possibly through the recruitment or stabilization of Ubp8p to SAGA.
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MATERIALS AND METHODS |
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Affinity purification of SAGA complexes.
The SAGA complex was purified from S. cerevisiae whole-cell extracts by using two different methods. First, the SAGA complex was immunoaffinity purified from wild-type (strain BY4743) and sgf11 deletion (strain 32781) strains by using polyclonal antibodies against Gcn5p as previously described (41). Second, tandem affinity purification (TAP) was used to purify Sgf11p as previously described (46). Basically, the TAP-SGF11 strain (7503040) was grown in 4 liters of YPD to an optical density at 600 nm of 1.0, and cells were harvested by centrifugation (
60 g wet weight). Cells were lysed with 0.5-mm zirconia-silica beads in lysis buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40, 1x Complete protease inhibitor [Roche]). The protein lysate was incubated with immunoglobulin G (IgG)-Sepharose beads (Pharmacia) for 1 h at 4°C and then poured into a disposable column. The beads were washed with lysis buffer, equilibrated with tobacco etch virus (TEV) cleavage buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 1.0 mM dithiothreitol [DTT]), and incubated with 5 U of TEV protease (Invitrogen) for 2 h at room temperature. After TEV cleavage, the IgG column eluate was incubated with calmodulin-affinity resin (Stratagene) for 1 h at 4°C, poured into a disposable column, and washed with calmodulin binding buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40, 1 mM imidazole, 2 mM CaCl2, 10 mM 2-mercaptoethanol). The bound proteins were eluted with calmodulin elution buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40, 1 mM imidazole, 20 mM EGTA, 10 mM 2-mercaptoethanol) and concentrated by trichloroacetic acid (TCA) precipitation. Purified protein complexes were reduced, alkylated, and digested with trypsin prior to analysis by mass spectrometry.
Mass spectrometry analysis of protein complexes. The DALPC mass spectrometry approach was used to identify purified proteins as previously described (29).
Immunoaffinity copurification assays.
The sgf11 deletion strain (32781) was transformed with the pYES-DEST52 vector or pYES-SGF11 and cultured overnight at 30°C in 50 ml of SC-Ura medium with 2% raffinose as a carbon source. SGF11 expression was induced by addition of galactose to a final concentration of 2%, and the yeast strains were cultured for an additional 6 h at 30°C. Cells were pelleted and resuspended in 1 ml of lysis buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5 mM 2-mercaptoethanol). Cells were lysed by using an equal volume of 0.5-mm zirconia-silica beads and a Mini-Beadbeater-8 (Biospec Products). Lysates were centrifuged at 20,000 x g for 15 min to remove particulate material. V5 epitope-tagged Sgf11p and endogenous Spt7p and Gcn5p were immunoaffinity purified from lysates by overnight incubation with either 2 µg of monoclonal anti-V5 (Invitrogen) coupled to 10 µl of protein A-Sepharose beads (Sigma) or 20 µg of affinity purified polyclonal anti-Spt7p or anti-Gcn5p antibodies (41) coupled to 10 µl of protein A-Sepharose beads. Beads were washed four times with 1 ml of wash buffer (20 mM HEPES [pH 7.9], 300 mM potassium acetate, 10% glycerol, 1 mM DTT, 1x Complete protease inhibitors). Proteins were eluted from the beads with Laemmli sodium dodecyl sulfate (SDS) sample buffer and separated by NuPAGE SDS-12% polyacrylamide gel electrophoresis (PAGE) and morpholineethanesulfonic acid buffer (Invitrogen) prior to immunoblot analysis.
Immunoblot analysis. NuPAGE gels were transferred to nitrocellulose membranes and blocked overnight in Tris-buffered saline containing 0.05% Tween and 5% nonfat dry milk. For immunoaffinity copurification assays, membranes were probed with either affinity-purified polyclonal antibody to Spt7p or monoclonal anti-V5 (Invitrogen). For SAGA composition assays, membranes were probed with an affinity-purified polyclonal antibody to Ubp8p, Tra1p, Taf12p, Gcn5p, and Taf10p (41). For HAT assays, Gcn5p was detected by using an affinity-purified polyclonal antibody (41). Membranes were washed five times in Tris-buffered saline containing 0.05% Tween and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Promega). Target proteins were visualized by autoradiography by using ECL Plus reagent (Amersham).
DNA microarray experiments.
Wild-type (BY4743) and sgf11 deletion (32781) strains were grown to an optical density at 600 nm of 1.0 in YPD at 30°C. Total RNA was extracted by using TRI Reagent and the manufacturer's suggested protocol (Molecular Research Center). Total RNA (100 µg) from each strain was incubated with 5 U of DNase (Promega) for 1 h at room temperature. The DNase was inactivated by incubation with 1 mM EDTA at 65°C for 10 min. The DNase-treated RNA samples were used for reverse transcription (RT) reactions. Oligo(dT)16 primer (2 µg) was added to the RNA sample, followed by incubation at 70°C for 10 min and chilling on ice for 5 min. The 30-µl samples were incubated with 30 µl of a labeling mixture (6 µl of dGTP-dATP-dCTP (each nucleotide at 1 mM), 2 µl of dTTP (1 mM), 4 µl of dUTP (1 mM), 12 µl of 5x first-strand buffer, 6 µl of DTT [0.1 M]), and 400 U of SuperScript II reverse transcriptase (Invitrogen) at 42°C for 2 h. RNA templates were removed by hydrolysis by first incubation at 95°C for 2 min and then incubation with 10 µl of 1 M NaOH and 10 µl of 0.5 M EDTA at 65°C for 30 min. After hydrolysis, 25 µl of 1 M HEPES (pH 7.5) was added, and cDNAs were purified by using a QIAquick kit (QIAGEN). The cDNAs were coupled to monofunctional NHS-ester Cy-dyes (Amersham). Wild-type cDNAs were labeled with Cy3 and cDNAs from the sgf11 deletion strain were labeled with Cy5. The dyes were resuspended in 10 µl of dimethyl sulfoxide, and 1.25 µl of the appropriate dye was added to each sample and incubated in the dark at room temperature for 1 h. The reactions were terminated by adding 4.5 µl of 4 M hydroxylamine (Sigma) and incubating them in the dark at room temperature for 15 min. The samples were combined, and the labeled cDNAs were purified by using a QIAquick kit. The labeled cDNAs were hybridized to DNA microarrays comprised of 6,735 yeast genes spotted in duplicate at high density on 75-by-25-mm glass slides. DNA microarrays were made by using the yeast genome oligonucleotide set (Operon) by the Vanderbilt Microarray Shared Resource Center.
DNA microarray data analysis.
The yeast arrays were scanned, and intensity analysis was performed by using GenePix Pro software (Axon). Gene annotation and interpretation were performed by using the program GeneTraffic (Iobion Informatics LLC, La Jolla, Calif.). The raw data were normalized by using a LOWESS sub-grid global normalization method. The following specific criteria were used to obtain a list of genes that were considered to be significantly affected by the sgf11 deletion. Genes were reported if there was a >2-fold change up or down from two independent experiments, the fold change was consistent between both experiments, and the effect was observed for the duplicate gene spots on each array.
RT-PCR experiments.
RT-PCR was used to validate selected microarray results. RNA isolated from wild-type (BY4743) and sgf11 deletion (32781) strains was reverse transcribed by using the protocol described for the microarray experiments. PCR was performed with the resulting cDNA and primer sets for CDC8 (5'-TCTGCCGCTAAGGGGACAAATG-3' and 5'-GCGCTTCAACTTCCTGAATGCC-3'), MAT
1 (5'-TTCGCAGCATCCTCCGCATTAG-3' and 5'-ACCAATGCCAAGCTTCAGCCTC-3'), and TDH3 (5'-TCTTCCATCTTCGATGCTGCCG-3' and 5'-AGCCTTGGCAACGTGTTCAACC-3') (Sigma Genosys). RT-PCR products were separated by using a 6% polyacrylamide gel cast in 0.5x Tris-borate-EDTA buffer and visualized by using ethidium bromide staining. Identical experiments were also performed with RNA isolated from a ubp8 deletion strain (30809).
HAT assays. The HAT Gcn5p was immunoaffinity purified from wild-type (BY4743) and deletion strains for gcn5, ada3, and sgf11 (37285, 33534, and 32781, respectively). Overnight cultures of each strain were grown in 50 ml of YPD at 30°C. Cells were pelleted and lysed in 1 ml of lysis buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol) by using 1 ml of 0.5-mm zirconia-silica beads and a Mini-Beadbeater-8. Lysates were centrifuged at 20,000 x g for 10 min to remove particulate material. Lysates were then incubated overnight at 4°C with 20 µg of affinity-purified polyclonal anti-Gcn5p IgG covalently coupled to protein A-Sepharose beads. After incubations, beads were washed three times with 1 ml of buffer (20 mM HEPES [pH 7.9], 300 mM potassium acetate, 10% glycerol, 1 mM DTT, 1x Complete protease inhibitor) and then washed with 1 ml of HAT buffer (75 mM Tris [pH 8], 50 mM NaCl, 0.1 mM EDTA, 1 mM MgCl2, 25% glycerol, 1 mM DTT, 5 mM sodium butyrate). For the HAT reaction, the washed beads were resuspended in 30 µl of HAT buffer containing 0.25 µCi of 3H-acetyl-CoA (ICN) and 5 µg of oligonucleosomes. The oligonucleosomes were prepared by using previously described methods (33). Reaction mixtures were incubated for 1 h at 30°C. Reactions were terminated with Laemmli SDS sample buffer. Aliquots (10 µl) were used for an anti-Gcn5p immunoblot analysis, and the proteins in the remaining sample were separated by NuPAGE on 4 to 12% gradient SDS-PAGE gels. To detect the quantity and migration of histones, gels were stained with R-250 Coomassie blue. The stained gels were incubated with ENHANCE (Perkin-Elmer Life Sciences), and histone acetylation was visualized by autoradiography.
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RESULTS |
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deletion strains by Gcn5p immunoaffinity purification in duplicate experiments. The components were analyzed by DALPC mass spectrometry. A comparative analysis was used to identify differences in SAGA components purified from the two strains (Table 2). The SAGA components with differences >1 standard deviation (SD) from the ratio of the estimated relative abundances in wild-type and sgf11 deletion strains were consider significant. These proteins were further examined by immunoblot analysis. The most dramatic observation from the mass spectrometry analysis was the absence of detectable Ubp8p in SAGA purified from the sgf11 strain (Table 2). A separate immunoblot analysis showed essentially equal amounts of Ubp8p in wild-type and sgf11 lysates. However, immunoaffinity-purified SAGA complexes from the two strains showed an 4-fold reduction of Ubp8p in SAGA purified from the sgf11 strain (Fig. 4A). The mass spectrometry analysis also showed differences of >1 SD for Taf12p and Gcn5p (Table 2). Differences for Taf12p and Gcn5p, as well as smaller differences observed for Tra1p and Taf10p, were examined by immunoblot analysis. Immunoblot analysis failed to detect significant changes in the amounts of Taf12p, Gcn5p, Tra1p, and Taf10p in SAGA purified from wild-type and sgf11 strains (Fig. 4B). Together, these results indicate that Sgf11p does not have a substantial role in maintaining the overall composition or integrity of SAGA but appears to mediate either recruitment or stable association of Ubp8p in SAGA.
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, ada3, and sgf11 deletion mutants by Gcn5p immunoaffinity purification. Purified complexes were incubated with purified oligonucleosomes and 3H-acetyl-CoA. After these reactions, histones were separated by SDS-PAGE and Coomassie blue stained, and acetylation was visualized with autoradiography. The primary band detected by autoradiography corresponds to the expected migration of histone H3 (Fig. 5). Immunoblot analysis was used to measure the amount of Gcn5p purified from each strain. As expected, Gcn5p was required for histone H3 acetylation. Gcn5p purification was less robust from the ada3 strain, and a reduced histone H3 acetylation was observed. These results are consistent with the requirement of Ada3p for SAGA-mediated HAT activity (1). In three independent experiments, Gcn5p purification and histone acetylation of the sgf11 deletion were similar to that of the wild-type strain. Together, these results suggest that the histone acetylation observed in our experiments is both Gcn5p/SAGA-dependent and Sgf11p independent.
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deletion strain and an isogenic wild-type strain. Genes were reported if there was a >2-fold change up or down from two independent experiments and the fold change was consistent between both experiments. The sgf11 deletion caused a >2-fold downregulation in the transcription of 37 genes and a >2-fold upregulation of 17 genes relative to the wild type (Tables 3 and 4, respectively). The values are averages from duplicate experiments. The raw data are available at http://www.linklab.mc.vanderbilt.edu. Although direct or indirect effects cannot be discriminated from these studies, comparing the effects of the sgf11 deletion to a previous microarray study for deletions of other SAGA components (27) suggests that SGF11 regulates the transcription of a subset of genes previously scored as SAGA dependent, as well as SAGA-independent genes.
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1, which was downregulated by the sgf11 deletion. MAT1, but not CDC8, was previously reported to be a SAGA-regulated gene (27). RNA was prepared from wild-type and sgf11 mutants independently from the samples used in the microarray experiments. The RT-PCR results for both genes were consistent with the DNA microarray experiments (Fig. 6). Based on these results for CDC8 and MAT1, we postulate that the genes listed in Tables 3 and 4 represent probable targets of Sgf11p-mediated transcription.
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deletion strain would have a pattern of gene transcription similar to the sgf11 deletion strain. To test this, we used RT-PCR to compare the mRNA expression levels of MAT1 in wild-type and a ubp8 deletion strain. Like the sgf11 deletion, deletion of ubp8 resulted in decreased expression of MAT1 mRNA relative to that of wild-type (Fig. 6). An increase in CDC8 expression in the ubp8 deletion strain was not observed (data not shown). Our results are consistent with a model in which SAGA-dependent transcriptional activation is regulated by Sgf11p through fostering an association of Ubp8p with SAGA. |
DISCUSSION |
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Role of Sgf11p in SAGA complex composition and HAT activity.
Reports have shown that the SAGA subunits Ada1p, Spt7p, and Spt20p function as scaffold or adaptor proteins and are required for the integrity of the SAGA complex (44, 48). Null mutants for these genes grow slowly when grown on low-quality carbon sources or when exposed to various transcription inhibitors (35, 44, 48). The generally robust growth of the sgf11 mutant is consistent with the fact that the SAGA complex is largely intact in the absence of Sgf11p. Moreover, SAGA composition analysis indicates that Sgf11p does not appear to have a substantial role in maintaining the integrity of SAGA but may be important for recruiting or retaining Ubp8p to SAGA.
An important aspect of SAGA function is its HAT activity. Studies with gcn5-null strains have demonstrated that GCN5-mediated histone acetylation regulates gene transcription in vivo (24, 25, 38). Other components of SAGA (Ada2p and Ada3p) are required for Gcn5p-mediated acetylation of nucleosomes in vivo (1) and for Gcn5p HAT activity in vitro (12). In contrast, Sgf11p is not required for Gcn5p-mediated acetylation of nucleosomal histone H3. By extension, we postulate that SAGA's HAT activity does not require Ubp8p, since this component is reduced in SAGA purified from the sgf11 deletion strain. In support of this postulation, it was recently reported that a ubp8 deletion does not alter SAGA-mediated HAT activity (6, 16).
Role of Sgf11p in SAGA-regulated transcription.
The importance of SAGA in regulating S. cerevisiae mRNA levels was demonstrated in a previous microarray study (27). By comparing transcript levels in wild-type and deletion mutants, the study showed that SPT20, GCN5, and SPT3 regulate 10, 4, and 3%, respectively, of the tested genes. Although some transcripts were affected by mutations in each of the three genes, others were affected by deletion of only specific, individual genes. The authors also propose that since other reports have shown deletion of spt20 results in a SAGA-null molecular phenotype (44), the genes regulated by spt20 deletion represent the totality of SAGA-dependent genes. Using DNA microarray analysis with similar growth conditions and applying similar statistical criteria, we show that deletion of sgf11 also affects transcription of a small subset of yeast genes (17 upregulated and 37 downregulated, 1% of the genes tested). Comparative analysis showed that 23 of 54 (43%) of the genes regulated by SGF11 were also regulated by SPT20 (Tables 3 and 4). Thus, SGF11 has a role not only in SAGA-dependent but also SAGA-independent transcription. Of the 268 reported GCN5 target genes, 9 are shared with SGF11, and only three of these are SPT20 regulated. Such a limited overlap of SGF11 and GCN5 target genes is expected, since sgf11 deletion has no apparent effect on Gcn5p association with SAGA or Gcn5p-mediated HAT activity. Of the common targets of SPT20 and SGF11, those that are regulated in the same direction are all reduced in the mutants. This suggests that Sgf11p largely promotes SAGA-mediated gene transcription. Ubp8p, whose association with SAGA is reduced in an sgf11 deletion strain, also regulates SAGA-activated genes (6, 16). We found that one of the genes affected by both SGF11 and SPT20, MAT1, is similarly affected by UBP8. Although the interaction of Sgf11p and Ubp8p was not examined further, these results suggest that the differences in SAGA-dependent gene activation in the sgf11 mutant are related to reduced association of Ubp8p with SAGA.
In addition to the core SAGA components, several other transcription regulators were also identified at low levels from the Sgf11p purification (data are available at http://www.linklab.mc.vanderbilt.edu). Although these interactions were not investigated further, interaction of Sgf11p with non-SAGA transcription regulators suggest a possible means by which SGF11 regulates SAGA-independent transcription. One identified protein of particular importance to SAGA-dependent transcription is TBP. Genetic studies have demonstrated that the SAGA components Spt3p and Spt8p regulate the binding of TBP to the TATA box of specific promoter and the activation of specific RNA polymerase II-dependent genes (2, 3). These data suggest a physical interaction between SAGA and TBP. In support of this, our clustering analysis indicates that SAGA and Sgf11p physically associate with TBP. Others have also shown that TBP copurifies from cell extracts with the SAGA components Ada3p and Spt20p (37, 39). From comparative analysis of our cDNA microarray results with those previously reported (27), we found that SPT3 and SGF11 share eight common gene targets, and six of these genes are also regulated by SPT20. Although further investigation is required, it is intriguing to speculate that common gene regulation by SPT20, SPT3, and SGF11 is a function of their interaction with and regulation of TBP.
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ACKNOWLEDGMENTS |
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This study was supported by an NIH grant ES11993 and NCI SPORE Lung Cancer Pilot Project Initiative awarded to A.J.L. (1 P50 CA90949). D.W.P was supported by NIH training grant T32-HL69765. C.M.W. and J.L.J. are supported by NIH grants GM64779 and HL68744. K.J.M. is supported by NIH grants ES11993, GM64779, NS43952, and GM68900. P.A.W. is supported by NIH grants ES11993 and GM52461. A.J.L. is supported by NIH grants ES11993, GM64779, HL68744, NS43952, and CA098131.
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FOOTNOTES |
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