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Vol. 16, Issue 6, 2605-2613, June 2005
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* Department of Biochemistry and Molecular Biology, University of California–San Francisco, San Francisco, CA 94143;
Department of Microbiology and Immunology, University of California–San Francisco, San Francisco, CA 94143
Submitted February 15, 2005;
Accepted March 7, 2005
Monitoring Editor: William Tansey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Green and Johnson, 2004). In other organisms, repression by Tup1-Ssn6 homologues is essential for cellular differentiation, specifically neurogenesis, hematopoiesis, and embryonic development, and a deeper understanding of the function of Tup1-Ssn6 in S. cerevisiae will likely shed light on the regulation of these diverse processes in other organisms (Pflugrad et al., 1997; Fisher and Caudy, 1998; Levanon et al., 1998; Chen and Courey, 2000). Tup1 is recruited to the genes it represses through an interaction with a sequence-specific DNA-binding protein responsible for the regulation of subsets of Tup1-repressed genes. Whereas the targets of the repressor complexes in fungi and metazoans depend on the specific needs of the organisms, it is believed the mechanism of Tup1-mediated transcriptional repression is conserved (Grbavec et al., 1999; Zhang and Emmons, 2002). Recent work suggests that Tup1-mediated repression is the result of the integrated contributions of distinct mechanisms (Lee et al., 2000; Bone and Roth, 2001; Wu et al., 2001; Zaman et al., 2001; Robyr et al., 2002; Green and Johnson, 2004; Zhang and Reese, 2004).
Several domains of Tup1 have been identified and are known to have varying effects on transcriptional repression (Komachi et al., 1994; Tzamarias and Struhl, 1994; Edmondson et al., 1996; Varanasi et al., 1996; Zhang et al., 2002). The highly conserved WD domain of Tup1 in particular has been shown to be sufficient for partial transcriptional repression. The well defined sequences of the degenerate WD repeat are found in many proteins of diverse functions and are typically sites of protein–protein interactions (Neer et al., 1994; Garcia-Higuera et al., 1996; Smith et al., 1999). The structural residues of the WD repeat necessary for folding are conserved, but much of the additional sequence in the repeat is varied, giving each WD protein a unique surface (Neer et al., 1994). Sprague et al. (2000) solved the crystal structure of the Tup1 WD domain (282–713aa of Tup1) and showed that this fragment assumed the propeller-like ring-shape typical of WD domains in other proteins (Figure 1A). In addition, these authors also described a high degree of amino acid conservation among fungi on one surface of the structure. This level of conservation suggested a vital, evolutionarily maintained function common to all fungal Tup1 homologues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Plasmid pAJ201 contains two LexA operators at the SmaI site in pLG
312S (Guarente and Mason, 1983). The a-specific gene reporter pKK78 contains three Mat
2 operator sites inserted into the SmaI site of pLG312S with the selectable marker switched to ADE2 and the 2µ sequences deleted to allow for integration. All LexA-fusion plasmids were made by inserting the full-length Tup1-LexA sequence (wild-type or mutated) and 800 base pairs (bp) of sequence upstream of the TUP1 open reading frame (ORF) into the NotI/XhoI site of pRS424 (Sikorski and Hieter, 1989). They are pSG82 (wild-type Tup1-LexA), pSG72 (Tup1F632A-LexA), pSG68 (Tup1R447A-LexA), pSG71 (Tup1D443A-LexA), pSG76 (Tup1Y489A-LexA), and pSG93 (Tup1E463A-LexA). Plasmid pSG96 has the 800-bp TUP1 upstream sequence fused directly to the LexA protein sequence.
Yeast Strains
The S. cerevisiae strains used in this study were all generated from a parental strain of genotype MAT ura3-52, lys2-801amb, ade2-101och, leu2-1, his3-200, trp1-1, which was descended from the original S288c strain. SGY84 (tup1) was constructed by transforming the parental S288c strain with a PCR product of the TRP1 gene flanked by homologous sequences of the TUP1 locus. SGY145 (tup1F632A), SGY146 (tup1R447A), SGY147 (tup1D443A), SGY148 (tup1Y489A), and SGY128 (tup1E463A) were made by transforming a full-length ORF fragment containing the mutation into a strain in which the TUP1 locus has been replaced with URA3, leaving 
200 bp of ORF homology on either side. Growth on 5-fluorootic acid selected for a strain in which the mutated TUP1 ORF had been integrated at the TUP1 genomic locus. These strains and SGY84 were then crossed to the MATa strain matching the parental S288c strain (SGY69) and sporulated to generate MATa versions of each mutant (SGY141, SGY142, SGY143, SGY144, and SGY140, respectively). For some of the microarrays, SGY200 was used for tup1R447A, which differs from SGY146 only in having had the trp1-1 deletion restored. SGY146 and SGY200 both have doubling times of 100 min in YPD at 30°C, and we do not believe the differences at the TRP1 locus are significantly influencing our results under the growth conditions used in these experiments (our unpublished data). The wild-type strain used in the microarrays was either SGY70 (the parental MAT strain described above) or SGY92 in which the trp1-1 deletion was restored.
SGY212, SGY220, and SGY226 are MATa strains with pKK78 integrated at the ADE2 locus and wild-type TUP1, tup1R447A, and tup1 at the genomic TUP1 locus, respectively. SGY213, SGY221, and SGY227 are the matching MAT strains. SGY215 is SGY70 with pKK602 integrated at the ADE2 locus. SGY219 was made by selecting for the replacement of a tup1 with a full-length TUP1-LexA sequence as described above and then integrating pKK602. The mutant Tup1-LexA fusion strains used in the ChIP experiments (SGY282 [tup1F632A-LexA], SGY253 [tup1R447A-LexA], SGY283 [tup1D443A-LexA], SGY284 [tup1Y489A-LexA], SGY285 [tup1E463A-LexA]) were derived from SGY219. A wild-type MAT strain was transformed with pAJ201 and then this strain was transformed with the Tup1-lexA fusion plasmids to make the strains used in the liquid 
-galactosidase assays: SGY286 (pSG96), SGY287 (pSG82), SGY288 (pSG72), SGY289 (pSG68), SGY290 (pSG71), SGY291 (pSG76), and SGY292 (pSG93).
Microarrays and Significance Analysis of Microarrays (SAM) Analysis
Microarrays of cDNA ORFs (6100 spots) were performed as described previously (http://derisilab.ucsf.edu/microarray/protocols.html; Green and Johnson, 2004). Each of the microarrays was done six times (from independently grown cultures), except for the tup1 and tup1R447A microarrays, which were done seven times (data available as Supplemental Material). The set of repeats for each strain was then analyzed by SAM using the one-class response and row average settings and the default random number seed (1234567). Seven hundred and twenty permutations (the complete set for six repeats) were done for all mutant data sets, except tup1 and tup1R447A, upon which 5000 permutations were performed. The delta values for all data sets were selected as the value that resulted in the lowest false discovery rate (FDR) calculated for the 90th percentile d-scores. Microarray and SAM analysis data for each Tup1 point mutant are included in Supplemental Tables 1 and 2.
Liquid -Galactosidase Assays
Quantitative assays were performed as described in Ausubel (1988). Activities are reported as Miller units and represent the average of measurements from three independently grown cultures for each strain.
Chromatin Immunoprecipitation and Quantitative PCR (QPCR)
Antibodies against LexA were purchased from Upstate Biotechnology (Lake Placid, NY) (catalog no. 06-719). Antibodies against Tup1 were generated using a bacterially expressed full-length Tup1-glutathione S-transferase (GST) fusion. Cultures were cross-linked with formaldehyde for 5 min, and ChIPs were performed with slight modifications as described previously (Strahl-Bolsinger et al., 1997). Extract from 50 to 100 ml of culture at OD600 1 was used for each immunoprecipitation. Extracts were sonicated seven times for 12 s by using a Branson sonifier 450 at 50% output power. ChIPs were analyzed by QPCR in a DNA Engine Opticon machine (MJ Research, Watertown, MA). PCR products were between 200 and 400 bp.
For a given Tup1 ChIP experiment, a median input ratio was calculated for each mutant strain versus wild-type -cells to normalize the amount of total DNA added to each immunoprecipitation (IP). The amount of immunoprecipitated DNA in each IP was normalized for input and the measurements from a and strains for each mutant were averaged. This average was then divided by the average of the measurements for tup1 a and strains to produce a relative level of enrichment for each mutant. QPCR reactions were done one to three times for each monitored genomic location from an individual ChIP experiment. Enrichment levels for each site are averages of data from three independent Tup1 ChIPs. For the Tup1 ChIP experiments analyzing the a-specific reporter, the ratios of QPCR measurements for the a and cells for the wild-type and tup1R447A strains (rather than the averages) were determined after normalization for input. The displayed data represents the average of these ratios from two independent ChIPs with three QPCRs performed for each ChIP.
The LexA ChIP experiments were performed as described above, with a median input ratio calculated for each strain compared with SGY215 (no LexA strain). The displayed data represents the average of the ratios of each strain to SGY215 from two independent ChIPs with three QPCRs performed for each ChIP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), the mapping of other fungal Tup1 homologues onto this structure revealed a strikingly conserved surface on one side of the propeller. When sequences of Tup1 homologues from more divergent organisms (Drosophila, Xenopus, Caenorhabditis elegans, and mice) were added to this alignment much of the conservation at this surface was maintained (our unpublished data). It seems likely that this surface of Tup1 is involved in a highly conserved set of interactions, and in this study we tested the functions of this surface in S. cerevisiae. We selected amino acids in the conserved surface of S. cerevisiae for mutation based on three criteria: 1) they are conserved in our expanded alignment of metazoan homologues of Tup1; 2) they are solvent exposed, and, as judged by the crystal structure, are not required for maintaining the structure of the protein; and 3) they are amino acids that are over-represented at sites of protein–protein interactions, as determined in a study of published mutational analyses (Bogan and Thorn, 1998). We chose five residues of Tup1 for mutagenesis to alanine, shown for convenience on a single molecule in Figure 1A. We constructed isogenic strains that had the individual mutant alleles of TUP1 integrated at the genomic TUP1 locus and verified that each mutant protein was stable and expressed at levels comparable to that of the wild-type Tup1 protein (Figure 1B). A region of Tup1 necessary for interaction with Ssn6 and complex formation has been localized outside of the domain used in the crystallization experiments that informed our selection of candidate residues for mutagenesis (Tzamarias and Struhl, 1994; Varanasi et al., 1996). From this observation and preliminary biochemical experiments that show our mutants interacting with Ssn6, we conclude that these five point mutants do not disrupt Tup1-Ssn6 complex formation (our unpublished data).
Microarray Analysis of Tup1 Point Mutants
To understand the impact of these point mutants on Tup1-mediated repression, we analyzed globally each of the mutant strains using expression microarray analysis. For each point mutant and an isogenic wild-type strain, we carried out six independent microarray hybridizations and analyzed the data by using SAM methodology (Tusher et al., 2001). In brief, SAM ranks each gene based on the magnitude of the change in expression and the reproducibility of that measurement in duplicate experiments. SAM also estimates the number of false positives (FDR) in a set of genes corresponding to a selected threshold of significance. We chose a threshold of significance for each of our data sets that yielded the lowest FDR for that data set, thus representing the most stringently selected set of regulated genes. Figure 2A lists the total numbers of significantly regulated genes in each of the Tup1 point mutant data sets. Each set typically has less than one predicted false positive and, as expected for mutations affecting transcriptional repression, the overwhelming majority (>93%) of the genes in each set are up-regulated, implying a loss of negative regulation. The few genes whose expression levels decrease in the point mutants generally show small magnitudes of changes in expression, and we believe this likely reflects indirect effects of Tup1-mediated repression.
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). Using these gene sets, we first determined the degree of overlap between the data sets of the point mutants. tup1R447A showed the strongest effects (the largest set of derepressed genes and the highest levels of derepression), and we saw significant overlap of all the other point mutant data sets with the set of genes derepressed by tup1R447A (Figure 2B). Moreover, there was considerable overlap between the data sets from any two of the other mutants (our unpublished data). The weakest effects overall in the magnitude of derepression are seen in the tup1D443A mutant, consistent with the small number of genes affected overall in this mutant. These findings suggest the five mutations can be arranged in a hierarchy and that they share at least one common defect but to varying degrees. The set of Tup1-repressed genes can be divided into four categories based on the effects of the mutants on their expression (Figure 2C). About half of the Tup1-repressed genes (53%) maintain wild-type levels of repression in each of the point mutant strains, as measured by microarray analysis. A smaller subset of genes is derepressed to some degree in all of the point mutants. A third set of genes is derepressed in the tup1R447A, tup1Y489A, and tup1E463A mutant strains but is unaffected by the other two point mutants. Finally, a fourth group of Tup1-repressed genes is significantly derepressed only in the tup1R447A mutant. In general, the magnitudes of changes in expression caused by the point mutants are less than that observed when TUP1 is deleted, suggesting that even at affected promoters the Tup1 point mutants are partially functional. However, one set of genes, the a-specific genes (and their downstream targets), is fully derepressed in the tup1R447A, tup1Y489A, and tup1E463A mutant strains (Figure 2C).
Tup1 Point Mutants Are Properly Recruited to Derepressed Genes
To accurately regulate the full set of its target genes, Tup1 must be recruited to the proper promoters. Point mutants that have a defect in Tup1-mediated repression may reflect 1) the inability of the Tup1 mutant to interact with its sequence-specific DNA-binding partners (a defect in recruitment to promoters) or 2) an inability to interact with the repression machinery once recruited to a regulated promoter. To test whether the repression defects of the Tup1 point mutants are due to a defect in recruitment, we used ChIP experiments to monitor the presence of Tup1 at regulated promoters in a wild-type strain and in the Tup1 mutant strains. We selected genes that are derepressed to varying degrees by the point mutants (Figure 3A). SUC2 is a previously known direct target of Tup1-mediated repression that was identified by SAM as significantly derepressed in only the tup1 and tup1R447A data sets. HSP12 and SPI1 are newly identified Tup1-controlled genes that were identified as derepressed by all five of the point mutants in our SAM analysis.
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Using antibodies to Tup1, we immunoprecipitated DNA from a wild-type strain, a tup1 strain, and the five Tup1 point mutants and measured the amount of precipitated DNA by quantitative PCR (QPCR). The ChIP from the tup1 strain, in which no Tup1 protein is expressed, measures the background (nonspecific) precipitation of DNA by the Tup1 antibodies and serves as our baseline against which to compare the amount of DNA precipitation in the other Tup1 strains. As expected, wild-type Tup1 occupies the three promoters (SUC2, HSP12, and SPI1) well above the background levels determined in the tup1 strain (Figure 3B). Because HSP12 and SPI1 had not been previously shown to be direct targets of Tup1-mediated repression, we measured the amount of Tup1 bound up- and downstream of the beginnings of the open reading frames. The enrichment of Tup1 occupancy at HSP12 and SPI1 is indeed focused at their promoters, and we conclude that these two genes are direct targets of Tup1-mediated repression. When analyzing the ChIP data for the Tup1 point mutants, we found that all five of the Tup1 point mutants also significantly occupied the three promoters and that overall the amounts of precipitated DNA were similar to that measured by ChIP in a wild-type Tup1 strain (Figure 3B). The enrichment of tup1R447A at SUC2 seems to be greater than that of wild-type Tup1, but we do not believe this is biologically significant. Regardless, this result shows that the defect in repression of tup1R447A at SUC2 is not due to a failure of the mutant protein to be recruited to the promoter.
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strain to those measured in our strongest mutant, tup1R447A (Figure 3C). For the most part, tup1R447A either had no effect on repression or only partially derepressed genes, the exception being the a-specific genes discussed below. However, there were a few fully derepressed genes that were not a-specific genes. ZRT1, a zinc transporter, was identified by SAM as a gene derepressed by tup1R447A and was derepressed to the same extent as that measured in a tup1 strain (median increases in expression over a wild-type strain of2.4- and 2.9, respectively). ChIP experiments measuring Tup1 recruitment to ZRT1 established that all of the Tup1 mutants showed the same levels of enrichment over background at the promoter as wild-type Tup1 (Figure 3D). Like HSP12 and SPI1, ZRT1 has not been shown to be a direct target of Tup1-mediated repression, so we confirmed that the ChIP signal we measured was concentrated at the ZRT1 promoter specifically (Figure 3D). This concentration at the promoter of ZRT1 is subtler than that seen for other genes we analyzed, but because the data displayed in Figure 3D comprises several independent repetitions of the experiments, we feel it is highly reproducible and represents a real phenomenon.
Tup1 Point Mutants Exhibit Repression Defects Even When Artificially Recruited to Promoters
The presence of the Tup1 point mutants at a fully derepressed gene implies that the mutations in Tup1 do not affect recruitment. To confirm that these mutant proteins exhibit a defect in repression independent of recruitment, we constructed LexA fusions of the Tup1 point mutants. In these strains, Tup1 is bound directly to a promoter containing a LexA operator via the fused LexA domain, bypassing the requirement for recruitment by a sequence-specific DNA-binding protein. We confirmed that the Tup1 mutant fusions (with the exception of tup1Y489A) could not repress a lacZ reporter that is under the control of LexA operators as efficiently as a wild-type Tup1-LexA fusion (Figure 4A). We then carried out ChIP experiments by using antibodies directed against LexA to establish the presence of the fusion proteins at the promoter of the lacZ reporter. All of the mutant Tup1 fusions were indeed bound to the reporter promoter, despite their inability to fully repress expression of lacZ (Figure 4B). As determined by QPCR, the amounts of DNA precipitated in the ChIP experiments were the same for the wild-type and mutant Tup1-LexA fusions. We performed ChIP experiments on these strains by using antibodies against Tup1 itself and obtained the same result (our unpublished data). Thus, although the LexA fusion proteins are efficiently bound to DNA, they are deficient in Tup1-mediated repression.
Some Mutations Can Disrupt Recruitment of Tup1 to the a-Specific Genes
We saw the strongest effects of the Tup1 point mutants on gene expression for one class of genes, the a-specific genes and their downstream targets. These genes were significantly derepressed only in the tup1R447A, tup1Y489A, and tup1E463A strains. Unlike the other subsets of Tup1-repressed genes, this group was typically fully derepressed compared with a deletion of TUP1. We confirmed this complete loss of repression when we quantitatively measured the level of repression in the tup1R447A mutant at a reporter construct repressed by Mat2, the sequence-specific DNA-binding protein regulating the a-specific genes. The a-specific genes are normally on in a cells, in which Mat2 is not present, and off in cells, which express Mat2. By comparing the levels of expression in and a cells (repressed vs. derepressed conditions) of a gene controlled by Mat2, we can determine the level of repression of that gene (Figure 5A, see figure legend). Figure 5A shows that tup1R447A is as defective in repressing an a-specific operator as a strain in which Tup1 is deleted (tup1). ChIP experiments showed that at this a-specific reporter, the Tup1R447A mutant was not properly recruited to the promoter in cells expressing Mat2 (Figure 5B). In contrast, Mat2, the sequence-specific DNA-binding protein responsible for recruiting Tup1, was properly bound to the a-specific reporter (our unpublished ChIP data), so we conclude that the absence of Tup1R447A from the reporter results from the inability of the mutant protein to interact with Mat2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, demonstrating that all of the mutant proteins are partially functional. Statistical analysis of expression microarray data for each mutant allowed us to rank the mutations based on the severity of their repression defects. tup1R447A emerged as the strongest mutant of Tup1 both in terms of the number of significantly derepressed genes and in the magnitude of the derepression at affected genes. The sets of significantly derepressed Tup1-regulated genes for the other mutants were almost entirely contained within the set of genes derepressed by tup1R447A. We conclude these mutations are disrupting a common aspect of Tup1-mediated repression.
Our global analysis of the repression defects proved much more informative than the use of a single Tup1-repressed gene or a reporter as the indicator of a loss of Tup1-mediated repression. By observing the effects of the Tup1 point mutants simultaneously on the complete set of Tup1-regulated genes, we were able to divide the larger group of genes into subsets based on their sensitivity to particular mutations of Tup1. A large portion (53%) of Tup1-repressed genes maintains full levels of repression, as determined by microarray analysis, in all of our mutant strains. The Tup1-regulated genes that are derepressed to some degree by one or more mutants can be placed into three subsets. One subset of genes is partially derepressed in all of the mutant strains. The genes in this subset do not seem to share any common functions or to be regulated by the same sequence-specific DNA-binding protein. A second subset of genes is derepressed by tup1R447A, tup1Y489A, and tup1E463A, but not by the other two mutants. Unlike the other subsets of Tup1-regulated genes, the subset affected by these three alleles is fully derepressed and is either directly or indirectly regulated by the sequence-specific DNA-binding protein Mat2. Finally, a third subset of Tup1-regulated genes is derepressed by only tup1R447A. These genes are only slightly derepressed compared with the magnitudes of changes in expression seen in a tup1 strain but are considered significant by SAM analysis. The genes in this subset do not seem to share a common functional or regulatory pattern; however, many are uncharacterized genes, and it is possible that a common theme will emerge in the future.
Further characterization of the point mutants revealed two distinct defects in Tup1-mediated repression. In the majority of the cases we tested, the point mutations disrupted the repression function of Tup1 but not its recruitment to DNA, as measured by ChIP experiments. This trend held for genes that were both partially and fully derepressed in the Tup1 mutant strains. Consistent with this interpretation, the Tup1 point mutants showed repression defects even when artificially recruited to promoters through fusion to LexA. ChIP experiments indicated that all of the mutant protein fusions occupied the LexA-binding site of a LacZ reporter, even though the mutant Tup1 fusion proteins could not repress transcription as effectively as a wild-type Tup1-LexA fusion.
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2, the sequence-specific DNA-binding protein that normally recruits Tup1, is clearly bound. We did not analyze tup1Y489A and tup1E463A in our ChIP experiments, but these two residues (Y489 and E463) were identified in a prior study of TUP1 designed to isolate mutations specifically disrupting the interaction between Tup1 and Mat2 (Komachi and Johnson, 1997). This earlier result, combined with our ChIP data and the observation that the a-specific genes are fully derepressed in the three mutants compared with levels seen in a tup1 strain, support the conclusion that these three residues of Tup1 are critical for the interaction between Tup1 and the sequence-specific DNA-binding protein Mat2. However, our microarray analyses of tup1R447A, tup1Y489A, and tup1E463A demonstrate that these three mutations also fail to fully repress Tup1-regulated genes outside of the group of a-specific genes, confirming that the mutations cause defects in repression beyond their inability to bind Mat2. These data indicate that two functions of Tup1—recruitment to a-specific gene promoters and transcriptional repression—both lie on this conserved surface of Tup1 and that the surfaces of the protein necessary for the two functions seem to overlap (Figure 6A). A mutant allele of the C. elegans homologue of Tup1 (unc-37) corresponds to a mutation of the E463 residue in the S. cerevisiae Tup1 and results in severe, pleiotropic effects, suggesting a severe loss of repression function (Pickles et al., 2002). The S. cerevisiae Tup1 crystal structure predicts a hydrogen bond interaction between the E463 and R447, the mutation of which results in the broadest, most severe defects discussed in this work. Thus, this surface of Tup1 is likely to carry out the same basic functions in diverse organisms.
Each Tup1–Ssn6 complex consists of four molecules of Tup1 and one molecule of Ssn6, so in the single Tup1 point mutant strains there would actually be four mutations per Tup1–Ssn6 complex formed (Varanasi et al., 1996; Redd et al., 1997). Therefore, a single point mutation could potentially disrupt four different interactions at a Tup1-repressed promoter. Based on this idea, it is not surprising that we see defects in multiple functions of Tup1 in our point mutant strains. For example, the same mutation could prevent the association of Tup1 with a DNA-binding protein, could disrupt interactions with components of the general transcriptional machinery, and could disrupt an interaction with a chromatin-modifying component (Figure 6B). Recent work has demonstrated that the full levels of repression at many Tup1-regulated genes requires contributions from several independent mechanisms, including mechanisms involving components of the transcriptional machinery as well as chromatin modifying factors (Smith and Johnson, 2000; Green and Johnson, 2004; Zhang and Reese, 2004). The relative contributions of these mechanisms to full transcriptional repression vary among Tup1-regulated genes, and these gene-specific requirements could explain the gradients of repression defects we observe. We believe that this surface of Tup1, which is conserved in Tup1 homologues from distantly related species, is critical for the orchestration of these multiple mechanisms of repression acting at a single promoter. The mutations described in this work will be valuable in working out additional details of the different mechanisms of Tup1-mediated repression and in understanding how those mechanisms work together to efficiently repress transcription in S. cerevisiae and other organisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Alexander D. Johnson (ajohnson{at}cgl.ucsf.edu).
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