Dual Regulation of the Met4 Transcription Factor by ... - Cell Press

Molecular Cell, Vol. 10, 69–80, July, 2002, Copyright 2002 by Cell Press

Dual Regulation of the Met4 Transcription Factor by Ubiquitin-Dependent Degradation and Inhibition of Promoter Recruitment Laurent Kuras,1 Astrid Rouillon,1 Traci Lee,2 Regine Barbey,1 Mike Tyers,2 and Dominique Thomas1 1 Centre de Ge´ne´tique Mole´culaire Centre National de la Recherche Scientifique 91198 Gif-sur-Yvettte France 2 Programme in Molecular Biology and Cancer Samuel Lunenfeld Research Institute Mount Sinai Hospital Toronto M5G 1X5 Canada

Summary The ubiquitin system has been recently implicated in various aspects of transcriptional regulation, including proteasome-dependent degradation of transcriptional activators. In yeast, the activator Met4 is inhibited by the SCFMet30 ubiquitin ligase, which recognizes and oligo-ubiquitylates Met4. Here, we demonstrate that in minimal media, Met4 is ubiquitylated and rapidly degraded in response to methionine excess, whereas in rich media, Met4 is oligo-ubiquitylated but remains stable. In the latter growth condition, oligo-ubiquitylated Met4 is not recruited to MET gene promoters, but is recruited to the SAM genes, which are required for production of S-adenosylmethionine, an unstable metabolite that is not present in rich medium. Thus, ubiquitylation not only regulates Met4 by distinct degradation-dependent and -independent mechanisms, but also controls differential recruitment of a single transcription factor to distinct promoters, thereby diversifying transcriptional activator specificity. Introduction The covalent modification of proteins by ubiquitin regulates a myriad of cellular processes (Hershko and Ciechanover, 1998). The best understood function of ubiquitylation is to target proteins for destruction by the 26S proteasome, a large compartmentalized protease particle that recognizes the polyubiquitin tag (Hochstrasser, 1996). More recently, nonproteolytic functions have been ascribed to ubiquitin conjugation, including targeting to different subcellular compartments and allosteric control of enzymatic events (Weissman, 2001). How ubiquitylation leads to such different responses in different contexts is not well understood. Ubiquitylation of a target protein is achieved through a cascade of E1, E2, and E3 enzymes, which activate and serially transfer ubiquitin to substrates (Hershko and Ciechanover, 1998). E3 enzymes, also called ubiquitin ligases, are the crucial determinants that select specific substrates for ubiquitylation. A recently described family of ubiquitin ligases, termed SCF complexes, regulates 1

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numerous cellular processes (Patton et al., 1998a). These multiprotein E3 complexes use substrate-specific adaptor subunits termed F box proteins to recruit substrates for ubiquitylation by a core apparatus, which is composed of the scaffold protein Cdc53/cullin, the RING finger protein Rbx1/Hrt1/Roc1, the adaptor protein Skp1, and the E2 enzyme Cdc34 (Koepp et al., 1999). Hundreds of proteins in sequence databases contain the ⵑ40 residue F box motif, which forms the binding site for Skp1 (Bai et al., 1996). In budding yeast, 22 different F box proteins can be identified, but only three of these have been studied in detail, namely Cdc4, Grr1, and Met30 (Patton et al., 1998b; Deshaies, 1999). SCFCdc4 and SCFGrr1 target the CDK inhibitor Sic1 and the G1 cyclins Cln1 and Cln2, respectively, and thus play a critical role at the G1-S transition (Feldman et al., 1997; Skowyra et al., 1997, 1999; Verma et al., 1997). The essential substrate of the SCFMet30 ubiquitin ligase is Met4, a transcriptional activator that regulates the MET gene network responsible for the biosynthesis of the sulfur-containing amino acids, methionine and cysteine (Patton et al., 1998b; Rouillon et al., 2000). Met4 functions within the context of multiprotein transcription complexes. Met4 contains an activation domain but is recruited to DNA by the cofactors Cbf1 and Met31/32, which bind two distinct promoter elements found in most Met4-responsive genes (Kuras et al., 1997; Blaiseau and Thomas, 1998). In addition to its role in the Met4 complex, Cbf1 also forms part of the centromere binding complex (Baker and Masison, 1990; Cai and Davis, 1990). Because Cbf1 is partly dispensable for MET gene expression, Met31/32 provide the main platform for recruitment of Met4 to DNA (Kuras and Thomas, 1995a; Blaiseau and Thomas, 1998). Upon exposure to high levels of methionine, yeast cells turn off MET gene expression through SCFMet30-dependent elimination of Met4 activity (Rouillon et al., 2000; Kaiser et al., 2000). In addition to regulation of methionine biosynthesis, Met4 also negatively regulates the G1-S transition by an unknown mechanism, such that cells lacking Met30 arrest in late G1 phase (Patton et al., 2000). The mechanism whereby SCFMet30 controls Met4 is controversial. In a previous study, we determined that addition of extracellular methionine triggers SCFMet30dependent ubiquitylation and proteasome-dependent degradation of Met4 (Rouillon et al., 2000). Subsequently, Kaiser et al. (2000) reported that Met4 ubiquitylation in response to excess methionine does not lead to Met4 degradation but rather results in the inhibition of Met4-dependent transcription. This inhibition was postulated to arise from the exclusion of Cbf1, but not Met4, from MET gene promoters. (Kaiser et al., 2000). In order to reconcile these apparently discrepant mechanisms of Met4 regulation, we systematically investigated the variables that might influence Met4 stability. Unexpectedly, the consequences of SCFMet30-mediated ubiquitylation of Met4 depended heavily on the cellular environment. When cells were grown in minimal medium, exposure to high methionine lead to rapid SCFMet30-dependent degradation of Met4 and corre-

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sponding depletion of Met4 from MET promoters. In contrast, when cells were grown in rich medium, oligoubiquitylated Met4 was stable and concentrated within the nucleus but was selectively excluded from MET promoters, whereas it remained bound to SAM gene promoters. These results indicate that distinct gene expression programs can be differentially regulated in response to ubiquitylation of a single transcription factor. Results Met4 Is Destabilized in Live Cells upon Methionine Addition Quantitative assessment of Met4 protein stability by immunodetection is complicated by several factors, including the use of epitope tags to detect Met4, extensive posttranslational modification of Met4 by ubiquitin, strain background, and the particular method of protein extraction (Kaiser et al., 2000; Rouillon et al., 2000). To circumvent the problem of protein extraction, we assayed Met4 abundance in living cells with a GFP-Met4 fusion protein. GFP-Met4 was expressed from the GAL1 promoter and its stability was monitored in a promoter shutoff experiment in the absence and presence of a high level of extracellular methionine (1 mM) in B minimal medium. The GFP-Met4 fluorescence signal was rapidly depleted, such that 80% of the signal disappeared within 60 min after promoter shutoff in the presence of methionine (Figure 1A). In contrast, the fluorescent signal remained constant upon promoter shutoff in the absence of methionine. As a control, a GFP-Met4⌬inh fusion protein, which is not recognized by SCFMet30 (Thomas et al., 1995), was stable even in the presence of methionine. To eliminate the possibility that heterologous expression of the GFP-Met4 fusion protein from the strong GAL1 promoter perturbed Met4 regulation, we replaced the wild-type chromosomal copy of the MET4 gene by a GFP-MET4 allele, in which the GFP moiety is inserted in-frame at nucleotide 45 of the MET4 reading frame. The fluorescent signal of GFP-MET4 cells grown in B medium was rapidly decreased by the addition of methionine, in a manner that depends on SCFMet30 activity (Figure 1B). GFP-Met4 regulates MET gene expression in a manner that is indistinguishable from wild-type Met4, and importantly, the kinetics of Met4 disappearance closely parallel loss of MET25 gene expression (Figure 1C). To corroborate the methionine-dependent elimination of Met4 in live cells, we determined the stability of an HA epitope-tagged version of Met4 expressed from the chromosomal locus. As expected, the HA3 Met4 protein was destabilized upon methionine addition to cells grown in B medium (Figure 1D). Met4 Occupancy at MET Promoters Is Decreased upon Methionine Addition The above results demonstrated that most Met4 was degraded upon addition of methionine to cells grown in minimal medium; however, it was still possible that a minor fraction of Met4 remained bound to promoter DNA and escaped degradation. To address this issue, we performed chromatin immunoprecipitation (ChIP) to analyze the abundance of Met4 directly at individual promoters. Moreover, to determine the correlation between

the presence of Met4 and transcriptional activity, we analyzed in parallel the association of the general transcription factor TFIIB with the same promoters. For this analysis, we used a strain in which the MET4 and SUA7 genes (encoding TFIIB) were replaced at the chromosome locus by epitope-tagged derivatives under control of the endogenous promoters (met4::HA3MET4 and sua7::SUA7MYC9, respectively). Crosslinked chromatin was prepared from cells grown in B medium before and 40 min after addition of 1 mM methionine. DNA associated with HA3Met4 and TFIIBMYC9 immune complexes was analyzed by quantitative polymerase chain reaction (PCR) with various primer pairs in the presence of radiolabeled 32P-dATP (Figure 2A). In the absence of methionine, HA3Met4 was heavily associated with the MET3, MET14, MET16, MET22, MET25, MET30, MMP1, and GSH1 gene promoters. In comparison, Met4 was only weakly associated with the unregulated control promoters of ADH1, GAL7, and tRNASer, or with a DNA sequence spanning the POL1 coding sequence. After addition of repressive amounts of methionine, Met4 was only weakly associated with the same Met4-dependent promoters. Quantitation of these results revealed that methionine addition resulted in a 4- to 25-fold decrease in Met4 occupancy at MET gene promoters (Figure 2B). There was also a small but reproducible decrease in levels of association with control sequences, probably due to the overall reduction of Met4 abundance. We note that regulated recruitment of Met4 binding to the MET30 promoter confirms the Met4-SCFMet30 negative feedback loop postulated previously (Rouillon et al., 2000). The identical chromatin preparations revealed a strong correlation between TFIIB and Met4 occupancy at MET gene promoters (Figure 2A). Thus, in accord with the decrease in Met4 occupancy, TFIIB occupancy decreased 3.3- to 7.3-fold upon addition of methionine (Figure 2B). Consistently, the decrease in TFIIB occupancy was followed by a parallel decrease in mRNA levels (Figure 2C). As expected, TFIIB occupancy at the Met4-independent ADH1 promoter was not altered by methionine. Finally, we ruled out the possibility that epitope tags and/or overexpression alter Met4 regulation by repeating these experiments with a polyclonal antibody raised against full-length recombinant Met4. This antibody specifically detected Met4 in wild-type cells, but not in met4⌬ cells (Figure 2D). Chromatin immunoprecipitations performed with this polyclonal anti-Met4 antibody recapitulated the above results, demonstrating that the presence of the HA epitope did not affect the behavior of Met4 at promoters (Figure 2E). Thus SCFMet30-mediated degradation of Met4 upon methionine exposure in minimal medium is accompanied by the disappearance of Met4 at biologically relevant promoter sequences. Met4 Is Present in Complete Medium Growing Cells Because these in vivo results were fully consistent with a Met4 degradation model (Rouillon et al., 2000), we sought to account for the discrepancy with the findings of Kaiser et al. (2000), which suggested a degradationindependent mechanism of Met4 regulation. One difference between the studies was the use of B minimal

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Figure 1. Destabilization of Gfp-Met4 Fusion Proteins in Living Cells Exposed to High Methionine Levels All cultures were grown in B minimal medium to early log phase in the presence of 0.2 mM of L-homocysteine as sulfur source. (A) Repression of ectopically expressed GFP-Met4. The GFP-Met4 (strain C300) and GFP-Met4⌬inh (strain C307) fusion proteins were expressed from the GAL1 promoter and then repressed by addition of glucose, either in the absence of methionine (⫺Met) or in the presence of 1 mM L-methionine (⫹Met). Fluorescence images were acquired at the indicated times. (B) Repression of endogenous GFP-Met4. met4::GFP-MET cells (strain CD240) and met4::GFP-MET, met30-2 cells (strain CY180-3B), both expressing the GFP-Met4 fusion protein from the endogenous MET4 promoter, were imaged at the indicated times after the addition (⫹Met) or not (⫺Met) of 1 mM L-methionine. (C) The MET gene network is regulated normally in cells that express GFP-Met4wt fusion protein in place of endogenous Met4. Total RNA was extracted from the strain C240 (met4::GFP-MET4) at the indicated times after addition of 1 mM L-methionine, and MET25 gene expression was assessed by Northern analysis. (D) met4::HA3MET cells (strain CD233) expressing the epitope-tagged HA3Met4 fusion protein from the endogenous MET4 promoter (see Experimental Procedures) were withdrawn at the indicated times after the addition (⫹Met) or not (⫺Met) of 1 mM L-met, and proteins were extracted using the TCA procedure (Rouillon et al., 2000) and processed for immunoblotting with monoclonal antibody to HA and polyclonal antibody to the yeast lysyl-tRNA synthetase (a generous gift of P. Kerjan).

medium versus either YNB or YPD medium. B medium is specifically designed to study sulfur metabolism and contains only essential amino acids, vitamins, and mineral salts, but no sulfur compounds (Cherest and SurdinKerjan, 1992). In contrast, YNB is a chemically defined complex medium that contains 40 mM ammonium sulfate, while YPD, sometimes called rich medium, contains yeast and bacterial extracts that contain high concentrations of undefined organic and inorganic sulfur sources (Sherman et al., 1979). Because sulfur metabolic fluxes are highly sensitive to growth conditions, such media differences might engender different regulatory responses (Thomas and Surdin-Kerjan, 1997). To test this hypothesis, we first examined whether different nutrient

conditions affected the stability of a GFP-Met4 fusion protein. In both B medium and YNB medium, high levels of extracellular methionine severely decreased the amount of GFP-Met4 signal (Figure 3A). The moderately lower level of GFP-Met4 signal in cells grown in YNB in the absence of methionine may be caused by the high concentration of ammonium sulfate in this medium. In contrast, in YPD medium, the GFP-Met4 signal was utterly insensitive to high extracellular concentrations of methionine. The quantitative assessment of steadystate levels of GFP-Met4 was corroborated by a qualitative kinetic analysis of GFP-Met4 disappearance upon methionine addition in each nutrient condition (Figure 3B).

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Figure 2. Met4p and TFIIB Occupancy at MET Promoters in Response to High Methionine Exposure (A) A strain expressing both HA3Met4 and TFIIB9MYC (CD269) or the untagged isogenic strain (W303-1A) was grown in B minimal medium as in Figure 1. Formaldehyde was added to the culture just before or 40 min after addition of 1 mM L-met, and the corresponding crosslinked chromatin preparations were immunoprecipitated with anti-HA or anti-Myc antibodies. Samples of the immunoprecipitated DNA and the total DNA used in the immunoprecipitation were amplified with primer pairs specific to the indicated Pol II promoters, the tRNASer(AGA) Pol III promoter, and the POL1 coding sequence (CDS). For each set of samples, the PCR products shown here derived from identical dilution and were checked to be in the linear range of the PCR reaction (data not shown). Immunoprecipitations were performed at least twice and yielded similar results. (B) Met4 and TFIIB occupancy at the indicated promoters were calculated from the experiment shown in (A) as described in Experimental Procedures. (C) Total RNA was extracted from the culture used in (A), and expression of the MET genes was quantified by Northern analysis. (D) Analysis of the specificity of the polyclonal antibody raised against Met4. Total proteins were extracted from wild-type (1: W303-1A) and met4⌬ (2: CC849-1B) cells and processed for immunoblotting with the anti-Met4 polyclonal antibody used at a 1:200 dilution. (E) A strain expressing untagged Met4 and TFIIB9MYC (CD268) and the isogenic untagged met4⌬ strain (CY231-7C) expressing no Met4 and untagged TFIIB were grown as described in (A). Crosslinked chromatin preparations were immunoprecipitated with antibodies against Met4 or the myc epitope. Immunoprecipated and the total DNA samples were amplified with primer pairs specific to the promoters indicated at the left.

Direct analysis of protein levels confirmed that in rich YPD medium, HA3Met4 is not degraded upon exposure of cells to high methionine (Figure 3C). Strikingly, HA3Met4 is highly modified in rich medium, as compared to B medium, regardless of methionine addition (Figure 3C).

Immunoprecipitation of HA3Met4 from cells grown in rich medium, either in the presence or absence of a Myctagged form of ubiquitin, confirmed that these highly modified forms correspond to Met4-ubiquitin conjugates (Figure 3D).


Figure 3. Quantification of GFP-Met4 Fusion Protein in Cells Grown in Different Media Cells expressing the GFP-Met4 fusion protein from the endogenous MET4 promoter (strain CD240) were grown in either B minimal medium, YNB minimal medium (drop-in), or YPD complete medium in the absence (⫺Met) or presence of 1 mM L-met (⫹Met). (A) For all growth conditions, the signal of 100 individual cells was recorded and quantified (see Experimental Procedures). (B) Cells grown either in YNB minimal medium or YPD rich medium were photographed at the indicated times after the addition (⫹Met) or not (⫺Met) of 1 mM L-met. (C) Cells (strain CD233) expressing the epitope-tagged HA3Met4 fusion protein from the endogenous MET4 promoter were grown in either YNB minimal medium or YPD rich medium. Proteins were extracted using the TCA procedure at the indicated times after the addition of 1 mM L-met and processed for immunoblotting with monoclonal antibody to HA and polyclonal antibody to the yeast lysyl-tRNA synthetase. (D) Ubiquitylation of HA3Met4 in rich medium; wild-type (W303-1A) cells containing a GAL1-HA3Met4-expressing plasmid and either a CUP1Myc Ub-expressing plasmid or an empty plasmid were grown overnight at 30⬚C in minimal medium in the presence of 2% raffinose media to an OD600 ⫽ 0.5. They were then filtered, washed with rich medium, suspended in prewarmed rich medium containing 2% galactose, and allowed to grow for 1 hr before harvesting. To induce the expression of the Myc-tagged ubiquitin, 100 ␮M of CuSO4 was added 2.5 hr before galactose induction. HA3Met4 was immunoprecipitated with anti-HA antibodies as described in Experimental Procedures. The samples were loaded on a 7.5% acrylamide gel and immunoblotted with anti-HA, anti-Myc, and anti-ubiquitin antibodies.

SCFMet30-Mediated Repression of MET Genes in Rich Medium The stability of Met4 in rich YPD medium was unexpected since it contains various organic sulfur sources, such that sulfate assimilation should be completely repressed, as should MET gene expression (Thomas and Surdin-Kerjan, 1997). Indeed, Northern analysis demonstrated that transcription of MET genes did not occur in YPD medium, even if Met4 was overexpressed from the strong GAL1 promoter (Figure 4A). Likewise, MET3, MET14, and MET16 were not expressed in wild-type cells grown in YPD medium (Figure 4B). An exception, however, was the MET25 gene, which was transcribed at approximately 20% of the level in cells grown in B medium. Therefore, in rich medium, Met4 is stable and nuclear localized yet unable to activate transcription of most of its target genes. The inability of Met4 to activate transcription of MET genes in rich medium depends on SCFMet30 activity, as revealed by Northern analysis of MET genes in met30⌬, cdc34-2, skp1-11, or cdc53-1 strains (Figures 4A and 4B). In contrast to wild-type cells, overexpression of MET4 from the GAL1 promoter in met30⌬ cells activated MET gene expression (Figure 4A). Likewise, Met4⌬inh,

which is insensitive to SCFMet30 (Thomas et al., 1995), also stimulated MET gene expression in rich medium (Figure 4B). Immunoprecipitation of HA3Met4 from met30⌬ mutant cells confirmed that Met4 is no longer ubiquitylated in rich medium growing cells lacking a functional SCFMet30 ubiquitin ligase (Figure 4C). Finally, a selenate resistance assay confirmed that the overall sulfate assimilation pathway is repressed through SCFMet30 function in rich medium. Whereas wild-type cells are resistant to this toxic sulfate analog in YPD medium, both met30-2 mutant cells or cells expressing Met4⌬inh are sensitive to selenate in rich medium (Figure 4D). Taken together, these results indicate that ubiquitylation inactivates Met4 in rich medium without affecting its stability. Loss of Met4 Occupancy at MET Promoters in Rich Medium To understand which aspect of Met4 activity is impaired by ubiquitylation in rich medium, we next analyzed both Met4 recruitment to promoter DNA and Met4-dependent transcriptional activation. The recruitment of both Met4 and TFIIB to MET gene promoters was assayed by chromatin immunoprecipitation, as described above. The

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Figure 4. SCFMet30 Mediated Repression of the MET Gene Network in Complete Medium Growing Cells (A) CC932-6D (met4::GAL1-MET4, MET30) and CC932-8B (met4::GAL1-MET4, met30::LEU2) cells were grown in complete medium containing raffinose as carbon source, and the expression of the GAL1-MET4 fusion gene was induced by transferring the cells in complete medium containing 2% galactose. Total RNA was extracted at the times indicated after the shift, and the expression of the GAL1-MET4, MET3, and MET16 genes was determined by Northern analysis. The actin probe was used as a control of the amount of RNA loaded. (B) The different strains were grown for eight generations in YPD complete medium at 28⬚C, half of the culture was shifted to 37⬚C for two hr, and then total RNA was extracted. Expression of MET genes was determined by Northern analysis. The actin probe was used as a control of the amount of RNA loaded. (C) HA3Met4 was immunoprecipitated as described in Figure 3D, except that met4⌬ met30⌬ cells (CC807-TL) were used and samples were immunoblotted with anti-HA, anti-Myc, and anti-ubiquitin antibodies. (D) W303-1A (wt), CC829-2B (MET4⌬inh), and CC975-2A (met30-2) were plated clockwise on YPD rich medium in the presence or absence of selenate (5 mM).

level of Met4 occupancy at Met4-dependent promoters was severely reduced in cells grown in YPD medium, as compared to cells grown in B medium lacking methionine (Figure 5A). Indeed, quantitative analysis indicated that the low level of promoter occupancy in rich medium was similar to that in B medium in the presence of methionine (Figure 5B). The degree of TFIIB occupancy at MET gene promoters was similarly reduced in rich medium (Figures 5A and 5B), in agreement with the lowlevel expression of MET genes under these conditions. These results suggest that SCFMet30-mediated ubiquitylation of Met4 abrogates its recruitment to most of its target promoters in cells grown in rich medium. To determine if Met4 transactivation activity per se is compromised in rich medium, we fused full-length Met4 to the LexA DNA binding domain and assessed the ability of the LexA-Met4 fusion to activate the transcription of a LexA operator-driven reporter gene. In contrast to endogenous Met4, the LexA-Met4 fusion protein is competent for transcriptional activation in both minimal and rich medium (Figure 5C). Importantly, immunoprecipitation assays revealed that the LexA-Met4 fusion protein is fully ubiquitylated in rich medium as the endogenous Met4 protein (Figure 5D). These results indicate that the defect in Met4 activity in rich medium is

due to a failure in promoter recruitment, perhaps through abrogation of interactions with one or more DNA binding factors within Met4 transcriptional complexes. MET Gene Repression in the Absence of Cbf1 In contrast to the above results, Kaiser et al. (2000) suggested that loss of Cbf1 from MET gene promoters, and not Met4, accounts for the repression of MET gene transcription. However, this argument is complicated by the fact that Cbf1 recruitment to MET promoter elements depends on another cofactor, Met28, and that the MET28 gene is itself regulated by Met4 (Kuras et al., 1997). Thus, a reduction in Cbf1 occupancy might well be an indirect consequence of attenuated Met4 activity. Meaningful assessment of the role of Cbf1 regulation by methionine necessarily requires that Cbf1 activity be decoupled from Met4 activity, for instance by heterologous expression of MET28. Since any such experiment would be in a nonphysiological context, we did not reexamine the association of Cbf1 with MET gene promoters. Instead, we tested a key prediction of the Kaiser et al. (2000) model, namely that regulation of MET genes by methionine should be abrogated in the absence of Cbf1. To do so, the kinetics of MET gene repression was examined in cbf1⌬ cells. Wild-type and cbf1⌬ mutant cells


Figure 5. Analysis of Met4 Binding Activity and Activation Capabilities in Cells Grown in Complete Medium (A) ChIP experiments were carried out on the HA3Met4, TFIIB9MYC cells (CD269) grown in minimal (with and without methionine) and in complete medium. Chromatin preparations were immunoprecipitated with anti-HA and anti-myc antibodies as described in Figure 2. Immunoprecipated and total DNA samples were analyzed by quantitative PCR with primer pairs specific to the indicated Pol II promoters and the POL1 coding sequence (CDS). (B) Quantification of the PCR products was conducted as in Figure 2. (C) Strain C190 (ura3::LexAop-XylE::URA3) was transformed by plasmids expressing either the LexAMet4-1 fusion protein or the LexA protein. Selected transformants were grown in either YNB minimal medium or YPD complete medium, and total RNA was extracted. The expression of the XylE reporter gene was determined by Northern assays. As a control, CD253 cells (leu2::MET16-XylE::LEU2) were grown in the same conditions, and the expression of the XylE reporter gene from the MET16 promoter was monitored by Northern. (D) The lexAMet4 fusion protein is ubiquitylated as the endogenous Met4; met4⌬ (CC849-TL) cells containing an ADH1-LexAMet4-expressing plasmid and either a CUP1-MycUb-expressing plasmid or an empty plasmid were grown overnight at 30⬚C in minimal medium and seeded into rich medium in the presence of 100 ␮M of CuSO4 to induce the CUP1 promoter. Cells were grown at 30⬚C to an OD600 ⫽ 0.5 before harvesting. The LexA-Met4 fusion protein was immunoprecipitated with anti-Met4 polyclonal antibodies, and the immunoprecipitated samples were immunoblotted with anti-Met4, anti-Myc, and anti-ubiquitin antibodies.

were grown in B minimal medium and assayed for expression of several MET genes upon addition of methionine. As expected from previous studies, MET genes are expressed at somewhat lower levels in cbf1⌬ cells than in wild-type cells (Kuras and Thomas, 1995a; O’Connell et al., 1995). However, it is also evident that transcription of the MET genes is repressed by methionine in both wild-type and cbf1⌬ cells (Figure 6). Thus, even if Cbf1 were actively excluded from MET gene

promoters, this mechanism would be insufficient to explain how oligo-ubiquitylation of Met4 by SCFMet30 represses transcriptional activation. Promoter Discrimination by Met4 in Rich Medium The above results explain the discrepancy between the behavior of Met4 in minimal versus rich medium, but at the same time pose a conundrum as to why the cell has evolved two separate control mechanisms, each

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Figure 6. Cbf1p Is Not Required for MET Gene Repression Wild-type (W303-1A) and cbf1⌬ (CC718-1A) strains were each grown in B minimal medium to early log phase (OD650 ⫽ 0.8) in the presence of 0.2 mM DL-homocysteine as sulfur source, shifted to B minimal medium without sulfur for 1 hr, and then a repressing amount of L-Met (1 mM) was added to the medium. Total RNA was extracted at the indicated times after L-Met addition, and expression of MET genes was determined by Northern analysis. The actin probe was used as a control of the amount of RNA loaded.

specific for different nutrient conditions. Specifically, why should oligo-ubiquitylated Met4 be stably maintained in the nucleus yet be unable to access its target promoters? Both metabolic studies (Dormer et al., 2000) and genome-wide DNA microarray analyses (P. Jorgensen, C. Peyraud, A. Bognar, T.L., D.T., and M.T., unpublished data) suggest that Met4 controls many genes in addition to those involved in sulfur amino acid biosynthesis. Beyond its function in protein synthesis, methionine serves as a precursor for the synthesis of S-adenosylmethionine (AdoMet), an important intermediary metabolite that is universally used as a methyl group

donor in transmethylation reactions of proteins, nucleic acids, and lipids and as a precursor in polyamine synthesis (Cantoni, 1977). Accordingly, genome sequence searches identified Met31/32 and Cbf1 binding elements, the AAACTGTG and TCACGTG core sequences, respectively, upstream of several AdoMet biosynthesis genes (Thomas and Surdin-Kerjan, 1997). To date, four genes involved in AdoMet metabolism have been identified in yeast, namely the SAM1, 2, 3, and 4 genes (Thomas et al., 2000). SAM1 and SAM2 are two highly related genes that encode the two yeast AdoMet synthase isoenzymes responsible for the synthesis of AdoMet from methionine. We therefore analyzed the binding of Met4 to the SAM gene promoters in cells grown in either minimal or rich medium. Contrary to most MET genes, Met4 recruitment to the SAM1/2 promoters was detected both in minimal medium lacking methionine and in rich medium (Figures 7A and 7B). Substantially less binding to the SAM1/2 promoters occurred in cells grown in minimal medium in the presence of high methionine concentrations. In the latter growth condition, repression occurs only after extracellular methionine has been converted into AdoMet, and thus transcription repression of both MET and SAM genes is in fact triggered by the increase of intracellular AdoMet concentration (Thomas et al., 1988). The regulation of the SAM2 gene evidently depends on more than Met4 recruitment, as recruitment of TFIIB only partially correlates with the presence of Met4 (Figure 7B). A phenotypic assay confirmed that SAM1 expression in rich medium is absolutely dependent upon Met4. The double met4⌬ sam2⌬ mutant is unable to grow in rich medium in the absence of exogenous added AdoMet, in contrast to both the single met4⌬ and the double met4⌬ sam1⌬ mutant cells, which are viable on unsupplemented rich medium (Figure 7C). Finally, Met4 could not be detected at the SAM4 promoter, consistent with its Met4-independent regulation (Thomas et al., 2000). Taken together, these findings suggest that in rich medium, SCFMet30-mediated ubiqui-

Figure 7. Met4p and TFIIB Occupancy at the SAM Promoters (A) Immunoprecipitated and total DNA samples from the ChIP experiment described in Figure 5 were amplified with primer pairs specific to the promoters of the SAM1, SAM2, and SAM3 genes. (B) Occupancy levels were calculated as in Figure 2. (C) CY283-7D (met4⌬), CY283-1B (met4⌬, sam1⌬), and CY283-14C (met4⌬, sam2⌬) were plated clockwise on YPD rich medium in the presence or absence of AdoMet (0.2 mM).


Table 1. Yeast Strains Strain

Genotype

Source

C190 C301 C307 CC718-1A CC807-1C CC807-TL CC823-4A CC849-1B CC849-TL CC932-6D CC932-8B CC975-2A CD233 CD240 CD253 CD268 CD269 CY180-3B CY231-7C CY283-1B CY283-14C CY283-7D MT1166 MT670 MT871 W303-1A

MATa, ade2, his3, leu2, trp1, ura3::LexAop-XylE::URA3 MAT␣, his3, leu2, trp1, met4::TRP1, ura3::GAL1-GFP-MET4::URA3 MAT␣, his3, leu2, trp1, met30-2, ura3::GAL1-GFP-MET4⌬inh::URA3 MATa, ade2, his3, leu2, trp1, ura3, cbf1::TRP1 MATa, ade2, his3, leu2, ura3, met4::TRP1, met30::LEU2 MATa, ade2, his3, leu2, ura3, met4::URA3, met30::LEU2 MAT␣, ade2, his3, leu2, trp1, ura3, MET4⌬inh MAT␣, ade2, his3, leu2, trp1, ura3, met4::TRP1 MAT␣, ade2, his3, leu2, trp1, ura3, met4::HIS3 MATa, ade2, his3, leu2, ura3, met4::GAL1-MET4 MATa, ade2, his3, leu2, ura3, met4::GAL1-MET4, met30::LEU2 MATa, ade2, his3, leu2, trp1, ura3, met30-2 MAT␣, his3, leu2, trp1, ura3, met4::HA3MET4 MAT␣, his3, leu2, trp1, ura3, met4::GFP-MET4 MATa, his3, trp1, ura3, leu2::MET16-XylE::LEU2 MAT␣, ade2, his3, leu2, trp1, ura3, SUA7-9MYC::TRP1 MAT␣, his3, leu2, trp1, ura3, met4::HA3MET4, sua7::SUA79MYC::TRP1 MAT␣, his3, leu2, trp1, ura3, met30-2, met4::GFP-MET4 MATa, ade2, his3, leu2, trp1, ura3, met4::TRP1, sua7::SUA79MYC::TRP1 ade2, his3, leu2, trp1, ura3, met4::TRP1, sam1::LEU2 ade2, his3, leu2, trp1, ura3, met4::TRP1, sam2::HIS3 ade2, his3, leu2, trp1, ura3, met4::TRP1 MATa, ade2, his3, leu2, trp1, ura3, skp1-11 MATa, ade2, his3, leu2, trp1, ura3, cdc34-2 MATa, ade2, his3, leu2, trp1, ura3, cdc53-1 MATa, ade2, his3, leu2, trp1, ura3

this study Rouillon et al., 2000 this study Kuras et al., 1996 Patton et al., 2000 this study this study Rouillon et al., 2000 this study Patton et al., 2000 Patton et al., 2000 this study this study this study P. Baudouin-Cornu this study this study this study this study this study this study this study Patton et al., 1998b Patton et al., 1998b Patton et al., 1998b R. Rothstein

tylation of Met4 allows selective repression of most MET genes while permitting expression of the SAM genes, most specifically SAM1, which in the absence of SAM2 is essential for AdoMet production in rich medium. Discussion Dual Regulation of the Met4 Transcription Factor by Ubiquitylation Ubiquitin-dependent regulation of Met4 by the SCFMet30 ubiquitin ligase has been reported to modulate Met4 transcription activity in response to excess methionine by degradation-dependent and -independent mechanisms (Rouillon et al., 2000; Kaiser et al., 2000). As previous studies differed substantially in experimental design, we reinvestigated potential regulatory mechanisms under carefully controlled growth conditions using more definitive experimental approaches, including use of a GFP-Met4 reporter to obviate complications of protein extraction, and use of an antibody that recognizes native levels of Met4 to eliminate possible artifacts caused by epitope tags. In minimal medium that lacks high-level sulfur sources, the bulk of GFP-Met4 is rapidly degraded upon methionine addition in a manner that depends on SCFMet30 activity. Correspondingly, the biologically relevant pool of Met4 at MET promoters is severely reduced, as are recruitment of the core transcriptional machinery and MET gene expression. In the course of these experiments, we found that the MET30 gene itself is a direct target of Met4, consistent with the negative feedback loop postulated to fine tune Met4 activity in response to fluctuations in methionine levels (Rouillon et al., 2000). SCFMet30-mediated degradation of Met4 thus accounts for all features of MET gene regulation in minimal medium.

In rich medium, regulation of Met4 activity is radically different from that in minimal medium. Under conditions of plentiful organic sulfur sources, Met4 is stable, oligoubiquitylated, and concentrated within the nucleus. However, despite the presence of Met4, most MET genes are not expressed under these conditions. Consistently, analysis of mRNA expression and selenate resistance demonstrate that SCFMet30 activity impairs the overall sulfur biosynthesis pathway in rich medium. The consequence of Met4 ubiquitylation by SCFMet30 evidently depends on environmental growth conditions. Unlike conventional substrates of the ubiquitin system, which are modified by extensive ubiquitin polymers, Met4 is merely oligo-ubiquitylated by the SCFMet30-Cdc34 complex (Kaiser et al., 2000). In the context of the SCFCdc4 and SCFGrr1 complexes, Cdc34 efficiently polyubiquitylates other substrates (Skowyra et al., 1997; Koepp et al., 1999), raising the intriguing question of how the extent of ubiquitin chain formation in the SCFMet30 complex is limited. In principle, the proteasome can recognize chains of four or more ubiquitin moieties (Deveraux et al., 1994; Thrower et al., 2000); however, it is unknown whether oligo-ubiquitylated forms of Met4 can be recognized by the proteasome. In point of fact, it is unclear whether Met4 is modified on a single site by an oligoubiquitin chain, as opposed to modification on multiple sites by single ubiquitin moieties. It is possible that Met4 degradation might require extension of Met4 ubiquitin conjugates into longer chain lengths, perhaps by an E4like activity (Koegl et al., 1999), which might respond to nutrient conditions. Ubiquitylation Impairs Met4 Recruitment to MET Promoters in Rich Medium To address the mechanism that inactivates Met4 in rich medium, we examined the biologically active pool of

Molecular Cell 78

Met4 at MET promoters by quantitative chromatin immunoprecipitation experiments. These experiments unequivocally demonstrated that Met4 does not occupy MET gene promoters in rich medium, thereby explaining the failure to recruit the RNA PolII subunit TFIIB and the failure to activate MET gene expression. Consistent with this mechanism, Met4 is nonetheless competent for transcriptional activation when it is tethered to promoter DNA by fusion to an ectopic DNA binding domain. It is possible that ubiquitylated forms of Met4 are unable to interact with one or more of the cognate DNA binding cofactors Met28, Met31, Met32, and Cbf1 (Blaiseau and Thomas, 1998). Because Met4 is the only component of these complexes able to provide transactivation function (Kuras and Thomas, 1995b), loss of Met4 is sufficient to explain the failure to recruit RNA PolII to promoter DNA. Consistent with this interpretation, it has been shown that oligo-ubiquitylated Met4 does not interact with Cbf1 in an extract system (Kaiser et al., 2000). Although Kaiser et al. (2000) have suggested that ubiquitylated forms of Met4 are retained at MET gene promoters with concomitant exclusion of the Met4 cofactor Cbf1, this model is at odds with most regulatory aspects of the Met4 system. In particular, Cbf1 is not essential for expression of MET genes (Kuras and Thomas, 1995a; P. Jorgensen, C. Peyraud, A. Bognar, T.L., D.T., and M.T., unpublished data), whereas the transactivation domain of Met4 is essential for expression (Kuras and Thomas, 1995b), and as show here, methionine addition represses MET gene expression even in a cbf1⌬ strain grown in minimal medium. As noted, it is possible that the loss of Cbf1 from MET gene promoters is an indirect consequence of Met28-dependent recruitment of Cbf1, coupled with Met4-dependent expression of MET28. Diversification of Transcription Factor Specificity by Ubiquitylation Both metabolic studies (Dormer et al., 2000) and genome-wide DNA microarrays analyses (P. Jorgensen, C. Peyraud, A. Bognar, T.L., D.T., and M.T., unpublished data) suggest that the Met4 regulon encompasses many more genes than those required for the sulfur amino acid biosynthesis. For example, Met4 controls a large cluster of 11 genes involved in oxidative metabolism (P. Jorgensen, C. Peyraud, A. Bognar, T.L., D.T., and M.T., unpublished data). Thus, while recruitment of Met4 to MET gene promoters is not necessary in rich medium, which contains organic sulfur compounds, Met4 may nevertheless be required for the activation of other genes. Indeed, we determined that the SAM genes constitute one set of genes whose expression relies on Met4 under rich nutrient conditions. SAM1 gene transcription in particular appears to be strictly Met4 dependent. The factors that recruit Met4 to the SAM promoters remain to be identified, but presumably such complexes are immune to interference by oligo-ubiquitylation of Met4. Intriguingly, it appears that the SWI-SNF chromatin remodeling machinery specifically derepresses MET genes in rich but not minimal medium (Sudarsanam et al., 2000). Thus it is possible that local chromatin remodeling effects might contribute to the selective exclusion of oligo-ubiquitylated forms of Met4 forms from different promoters in rich medium.

Regardless of the precise mechanism, the discrimination between different subsets of Met4 target genes may allow cells to fine tune metabolic flux in response to various nutrient conditions. In the case of the SAM genes, we note that AdoMet is a very unstable molecule that is not present in rich medium. Cells impaired in AdoMet synthesis, such as sam1⌬ sam2⌬ or met4⌬ sam2⌬ double mutants, are unable to grow on YPD medium in the absence of exogenous AdoMet (Thomas et al., 1988; this paper). This finding exemplifies how posttranslational modification such as ubiquitylation allows the differential recruitment of one transcription factor to target promoters, thereby engendering distinct gene regulations. This mechanism may be of quite general significance given the emerging links between the ubiquitin-proteasome system and the transcriptional machinery (Salghetti et al., 2001; Tansey, 2001). Experimental Procedures Yeast Strains and Media S. cerevisiae strains used in this study are listed in Table 1. B minimal medium is a synthetic medium that lacks organic and inorganic sulfur sources (Cherest and Surdin-Kerjan, 1992). Unless indicated, cells were grown in the presence of 0.2 mM DL-homocysteine as a sulfur source. YNB minimal medium contains 0.143% yeast nitrogen base (Invitrogen, Carlsbad, CA ), 0.5% ammonium sulfate, and 2% glucose. For both B and YNB minimal media, only the amino acids and bases required to complement the auxotrophic requirements of each strain were added (i.e., drop-in medium). YPD complete medium contains 0.5% yeast extract (Difco), 0.5% bacto-peptone (Difco), and 3% glucose. S. cerevisiae cells were transformed by the lithium acetate method (Gietz et al., 1992). Plasmid Construction To create the met4::GFP-MET allele in which the GFP moiety is inserted in-frame at nucleotide 45 within the MET4 chromosomal gene (numbered from the start codon), the GFP3 ORF (Cormack et al., 1996) was amplified by PCR using the Pfu polymerase (Promega) and cloned into the EcoRI site of plasmid pProMet4-1 (Kuras and Thomas, 1995b). The resulting plasmid, pProMet4GFP, contains 600 bp of the 5⬘ upstream region of MET4 and the first 45 bp of the MET4 ORF followed by the GFP gene. The pProMet4GFP plasmid was digested by EcoRI and BamHI and ligated with the EcoRIBamHI fragment of plasmid pLexMet4-1 (Kuras and Thomas, 1995b), which contains the entire MET4 gene except for the first 45 bp and 300 bp of the 3⬘ downstream region of MET4, to yield plasmid pGFPMET4wt. This plasmid was digested with XbaI to liberate a fragment containing the GFP-MET4 fusion together with the 5⬘ and 3⬘ MET4 flanking regions, which was used to transform CC849-8A cells harboring a met4::TRP1 disrupted allele. Correct replacement was ascertained both by Southern and PCR analyses. A met4::HA3MET4 allele in which three HA tags are inserted in-frame with the MET4 chromosomal gene was created in the same manner except that GFP sequences were replaced by three tandem HA epitopes. Protein and RNA Analysis Total proteins were extracted by glass bead lysis in buffer containing 1% deoxycholic acid, 1% Triton-X-100, 0.1% SDS, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 1 mM DTT, 5 mM NEM, 1 mM PMSF, 1 ␮g/ml leupeptin, and pepstatin. For immunoprecipitation of HA3Met4 tagged protein, 0.25 ␮g of 12CA5 mouse monoclonal anti-HA antibody was added to 5 mg of cell extract. For immunoprecipitation of the endogenous Met4 protein, 1 ␮l of anti-Met4 antibody was added to 5 mg of extract. Samples were incubated on ice for 1 hr, bound to 30 ␮l of 50% protein A bead slurry for 1 hr at 4⬚C, and washed four times with 1 ml lysis buffer each. Samples were separated on a 7.5% acrylamide gel, transferred to a PVDF mem-


brane, and probed with anti-Myc antibody (1:5,000 dilution), antiMet4 polyclonal antibody (1:200 of 5th bleed), or 12CA5 anti-HA antibody (1:10,000 dilution), followed by peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1: 10,000 dilution) and detected by SuperSignal West Pico chemiluminescent substrate (Pierce). Total cellular RNA was extracted from yeast by the hot phenol method, separated, and probed as described (Rouillon et al., 2000). Fluorescence Microscopy GFP-Met4p fusion protein signals were monitored in living cells on a Nikon Eclipse fluorescence microscope using an Omega XF116 filter. All images were collected with a Princeton CCD camera using identical settings and analyzed with the Meta-Imaging V4.5 software (Universal Imaging, Downingtown, PA). For each cell, the limits of the nucleus and of the full cell were delimited, the sizes (SN and SC, respectively) of the two areas were calculated, and the fluorescent intensities (IN and IC, respectively) of each area were recorded. The signal intensity IQ corresponding to the GFP-Met4 signal was calculated for each cell as IQ ⫽ IN ⫺ SN ⫻ ((IC ⫺ IN)/(SC ⫺ SN)). For each growth condition, the quantification [IQ] was the average of the IQ calculated for 100 different cells and expressed in arbitrary units. For some observations, when indicated, nuclei were stained using the dye HOECHST n⬚33342 (Sigma), which was added at 1␮g/ml to the culture 20 min prior to imaging. Chromatin Immunoprecipitation Crosslinked chromatin preparation and immunoprecipitation (ChIP) was performed as described previously (Kuras and Struhl, 1999), except that all immunoprecipitations were carried out in the presence of 150 mM NaCl instead of 275 mM NaCl. The antibodies used are the following: the mouse monoclonal HA antibody F-7 from Santa Cruz, the mouse monoclonal Myc antibody PL14 from StressGen, and a rabbit polyclonal Met4 antiserum produced against fulllength Met4 produced in insect cells. Immunoprecipitated and total DNA samples were analyzed by quantitative PCR in the presence of [␣-P32]dATP as described previously (Kuras and Struhl, 1999). Linearity of the PCR was assayed by testing in parallel multiple independent dilutions of each sample. PCR products (typically 200– 300 bp) were separated on an 8% TBE polyacrylamide gel and quantified by PhosphorImager analysis (Molecular Dynamics). The occupancy level at a given promoter is defined as the ratio of immunoprecipitated DNA over total DNA for each PCR product. The occupancy level at MET25 was arbitrarily set to 100, and all other values were represented relative to this standard. Acknowledgments We thank Yolande Surdin-Kerjan for her constant support and helpful discussions. This work was supported by the Centre National de la Recherche Scientifique and the Association de la Recherche sur le Cancer. M.T. is supported by the National Cancer Institute of Canada, the Canadian Institutes of Health Research, and the Human Frontiers Science Programme and holds a Canada Research Chair in Biochemistry. A.R. is supported by a thesis fellowship from the Fondation de la Recherche Me´dicale. Received: February 19, 2002 Revised: May 7, 2002

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