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<?xml version="1.0" ?> <tei> <teiHeader> <fileDesc xml:id="0"/> </teiHeader> <text xml:lang="en"> <front>© 2005 Nature Publishing Group <lb/>A putative stimulatory role for activator turnover in <lb/>gene expression <lb/>J. Russell Lipford 1 , Geoffrey T. Smith 1 , Yong Chi 1 † & Raymond J. Deshaies 1 <lb/> The ubiquitin–proteasome system (UPS) promotes the destruc-<lb/>tion of target proteins by attaching to them a ubiquitin chain that <lb/>is recognized by the 26S proteasome 1 . The UPS influences most <lb/>cellular processes, and its targets include transcriptional activa-<lb/>tors that are primary determinants of gene expression. Emerging <lb/>evidence indicates that non-proteolytic functions of the UPS <lb/>might stimulate transcriptional activity 2,3 . Here we show that <lb/>the proteolysis of some transcriptional activators by the UPS <lb/>can stimulate their function. We focused on the role of UPS-<lb/>dependent proteolysis in the function of inducible transcriptional <lb/>activators in yeast, and found that inhibition of the proteasome 4 <lb/>reduced transcription of the targets of the activators Gcn4, Gal4 <lb/>and Ino2/4. In addition, mutations in SCF Cdc4 , the ubiquitin <lb/>ligase for Gcn4 (ref. 5), or mutations in ubiquitin that prevent <lb/>degradation 6 , also impaired the transcription of Gcn4 targets. <lb/>These transcriptional defects were manifested despite the <lb/>enhanced abundance of Gcn4 on cognate promoters. Proteasome <lb/>inhibition also decreased the association of RNA polymerase II <lb/>with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCF Cdc4 <lb/>for Gcn4 targets. Expression of a stable phospho-site mutant of <lb/>Gcn4 (ref. 7) or disruption of the kinases that target Gcn4 for <lb/>turnover 5,7 alleviated the sensitivity of Gcn4 activity to defects in <lb/>the UPS. <lb/></front> <body>The UPS is a fundamental component of normal cell growth and <lb/>proliferation. The UPS is also important for cancer cell growth, as <lb/>highlighted by the recent approval of the proteasome inhibitor, <lb/>Velcade, for the treatment of relapsed multiple myeloma 8 . Recent <lb/>studies have investigated the mechanism of Velcade action by <lb/>examining the transcriptional response to the drug in human and <lb/>yeast cells 9,10 . These studies indicate that proteasome inhibition does <lb/>not substantially alter bulk transcription of the genome in myeloma <lb/>cells or in Saccharomyces cerevisiae. However, a group of genes are <lb/>repressed by Velcade, including human growth and survival genes <lb/>and yeast genes involved in the biosynthesis of amino acids 9,10 . These <lb/>yeast genes are regulated by the b-ZIP transcriptional activator Gcn4, <lb/>which promotes the expression of more than 500 genes 11 . Gcn4 is a <lb/>target for UPS-mediated degradation 12 through the E3-ubiquitin <lb/>ligase SCF Cdc4 . Ligases comprise the final, substrate recognition step <lb/>of the ubiquitination cascade. SCF Cdc4 ubiquitinates and targets for <lb/>proteolysis Gcn4 molecules that have been phosphorylated by the <lb/>cyclin-dependent kinases (CDKs) Srb10 and Pho85 (refs 5, 7). We <lb/>have attempted to explain the role of the UPS in the function of Gcn4 <lb/>and other activators. <lb/>To assess the impact of proteolysis on Gcn4 function, we treated a <lb/>yeast strain (pdr5D) that is sensitive to proteasome inhibitors 10 with <lb/>the Velcade analogue MG132 (ref. 4). Reverse transcriptase-mediated <lb/>polymerase chain reaction (RT–PCR) analyses confirmed that, as <lb/>for Velcade 10 , treatment with MG132 substantially reduced the <lb/>transcription of several Gcn4 targets, including HIS4 and CPA2, in <lb/>minimal medium, in comparison with dimethylsulphoxide (DMSO) <lb/>alone (Fig. 1a and Supplementary Fig. S1a). Similar results were <lb/>obtained when Gcn4 was highly induced by amino acid starvation <lb/>(Fig. 1a, 2Leu; see Supplementary Information for discussion of <lb/>media). These effects were largely dependent on Gcn4, because we <lb/>observed similar decreases in the transcription of a reporter driven <lb/>exclusively by six Gcn4-binding sites 13 (Fig. 1a, GCRE6–LacZ). <lb/></body> <front>LETTERS <lb/></front> <body>Figure 1 | UPS-dependent proteolysis positively regulates inducible <lb/>transcriptional activators. a, Indicated strains were grown in minimal or <lb/>starvation (2Leu) medium, treated with MG132 (50 mM) or DMSO, and <lb/>processed for RT–PCR of the indicated transcripts. pdr5D enables the uptake <lb/>of MG132 into yeast. b, A pdr5D strain was induced with galactose, treated <lb/>with MG132 and prepared for RT–PCR of GAL1 and ACT1. c, wild-type <lb/>(WT) and pre1-1, pre4-1 strains were grown in minimal medium at 27 8C <lb/>and processed for RT–PCR of HIS4 and ACT1. d, Conditionally expressed <lb/>ubiquitin was depleted from strains while expression from a complementing <lb/>plasmid encoding either WT ubiquitin, no ubiquitin (D) or K48R-ubiquitin <lb/>was induced (see Supplementary Information). Samples were prepared for <lb/>RT–PCR as above. e, WT (white bars), cdc34-2 (grey bars) and gcn4D (black <lb/>bars) strains expressing LacZ from the HIS3 or GCRE6 promoter were <lb/>grown in minimal or starvation medium (3-AT) at 30 8C and then processed <lb/>to test b-galactosidase (b-gal) activity. Standard deviations are from three <lb/>replicates. n.d., not detectable. f, WT, cdc34-2 and cdc4-1 strains were grown <lb/>at 27 8C or shifted to 30 8C for 1 h. RT–PCR was performed for HIS4, ARG1 <lb/>and ACT1. <lb/></body> <front>1 Howard Hughes Medical Institute, Division of Biology, MC 156-29, California Institute of Technology, 1200 E. California Boulevard, Pasadena, California 91125, USA. <lb/> †Present address: Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103, USA. <lb/> Vol 438|3 November 2005|doi:10.1038/nature04098 <lb/> 113 <lb/></front> <note place="footnote">© 2005 Nature Publishing Group <lb/></note> <body>We also tested the proteasome dependence of two regulons (GAL <lb/>and INO) that might have eluded the Velcade microarray analysis <lb/>because they were not expressed under the growth conditions that <lb/>were employed. MG132 substantially decreased induction of the Gal4 <lb/>target, GAL1, on the addition of galactose (Fig. 1b). Similarly, on the <lb/>removal of inositol, transcription of INO1, a target of the activators <lb/>Ino2 and Ino4, was largely abrogated by MG132 (Supplementary <lb/>Fig. S1b). In all cases treatment with MG132 did not affect the <lb/>constitutive transcription of ACT1. <lb/>These findings indicate that the 26S proteasome might promote <lb/>the function of some inducible transcriptional activators. Focusing <lb/>on Gcn4, we tested additional components of the UPS for their <lb/>impact on transcription. A strain (pre1-1 and pre4-1) mutated for the <lb/>peptidase activity of the proteasome showed reduced transcription of <lb/>HIS4 in comparison with a wild-type (WT) strain (Fig. 1c) 14 . We also <lb/>tested transcription of HIS4 in strains that conditionally express <lb/>alternative versions of ubiquitin 6 . When endogenous ubiquitin was <lb/>depleted in the presence of a vector plasmid (UbD) or depleted in a <lb/>cell expressing K48R ubiquitin, HIS4 messenger RNA levels were <lb/>sharply decreased in comparison with a depleted strain that <lb/>expressed WT ubiquitin (Fig. 1d). Again, the ACT1 mRNA remained <lb/>constant. These findings confirm that ubiquitination and proteolysis <lb/>are important for Gcn4 function. Importantly, because the <lb/>K48R mutant cannot form chains that target substrates to the <lb/>proteasome, these findings also indicate that, in contrast to <lb/>the regulation proposed for Gal4–VP16 (ref. 15) and c-Myc 16,17 , <lb/>mono-ubiquitination might not be able to sustain Gcn4 activity. <lb/>We next examined the impact of Cdc34–SCF Cdc4 , the specific <lb/>E2–E3 ubiquitin ligase for Gcn4 (refs 5, 7), on activator function. <lb/>Gcn4-dependent expression of b-galactosidase was evaluated in WT, <lb/>temperature-sensitive cdc34-2, and gcn4D strains. After growth at the <lb/>semi-permissive temperature of 30 8C, the cdc34-2 strain exhibited a <lb/>fourfold decrease (relative to WT) in reporter expressed from the <lb/>HIS3 promoter (HIS3P) in minimal medium and a roughly 15-fold <lb/>reduction on starvation by 3-aminotriazole (3-AT, ref. 11) (Fig. 1e). <lb/>Expression from GCRE6–LacZ was also compromised about 10-fold <lb/>in cdc34-2 (Fig. 1e). In addition, expression from both promoters was <lb/>extinguished in gcn4D cells under all conditions (Fig. 1e). RT–PCR <lb/>analysis was then used to assess effects on endogenous targets. <lb/>Gcn4-dependent transcription of HIS4 and ARG1 was defective in <lb/>both cdc34-2 and cdc4-1 (a thermosensitive allele of the SCF F-box <lb/>protein Cdc4) strains at 30 8C (Fig. 1f). In contrast, ACT1 transcript <lb/>levels in all strains were similar. These results point to a stimulatory <lb/>role for Cdc34–SCF Cdc4 in Gcn4-mediated transcription. <lb/>To explore the molecular basis of the stimulation of Gcn4 function <lb/>by the UPS, we examined the effect that proteasome inhibition has on <lb/>the abundance and ubiquitination of Gcn4. Western blotting showed <lb/>that MG132 increased the abundance of chromosomally encoded <lb/>Gcn4–Myc9 and led to the appearance of a high-molecular-mass <lb/>ladder (Fig. 2a, lanes 1 and 2). Immunoprecipitation and western <lb/>analysis with anti-ubiquitin antibodies confirmed that this ladder <lb/>was ubiquitinated Gcn4 (Fig. 2a, lanes 5, 6, 9 and 10). Because <lb/>transcription mediated by Gcn4 was strongly repressed by MG132 <lb/>(Fig. 1), ubiquitination was presumably insufficient to sustain Gcn4 <lb/>activity. To confirm the specificity of Gcn4 ubiquitination, we <lb/>repeated the analysis with a gcn4–3T2S strain. Gcn4–3T2S lacks <lb/>five phosphorylation sites, is no longer phosphorylated by Srb10 or <lb/>Pho85 nor ubiquitinated by SCF Cdc4 in vitro, and is stabilized 7 . As <lb/>predicted, the 3T2S strain had much lower levels of ubiquitinated <lb/>Gcn4 (Fig. 2a, lanes 3, 4, 7, 8, 11 and 12). <lb/>We next investigated promoter occupancy by Gcn4 and Gal4 and <lb/>recruitment of RNA polymerase II (polII) to target genes. Chromatin <lb/>immunoprecipitation (ChIP) assays 18 revealed a substantial increase <lb/>in the association of Gcn4–Myc9 with the HIS4 promoter in cells <lb/>treated with MG132 (Fig. 2b, 9E10). Similar experiments showed <lb/>little change in levels of TAP-tagged Gal4 at the GAL1 promoter on <lb/>treatment with MG132 (Fig. 2c, IgG). The galactose-dependent <lb/>decrease in promoter-bound Gal80–TAP was also unaffected by <lb/>MG132 (Supplementary Fig. S3). When the ChIP was performed <lb/>with polII, MG132 reduced the signal for the promoter, open reading <lb/>frame (ORF) and terminator regions of the Gcn4 target HIS4, <lb/>whereas the signal for the ACT1 ORF was unaffected (Fig. 2b, <lb/>polII). MG132 also decreased the polII ChIP signal for the GAL1 <lb/>promoter and ORF (Fig. 2c, polII) and the INO1 promoter (data not <lb/>shown). <lb/>The ChIP analysis was extended to cdc34-2 and cdc4-1 strains. At <lb/>30 8C, more Gcn4–Myc9 (Fig. 2d, 9E10) but less polII was associated <lb/>with the HIS4 promoter in the SCF mutants than in the WT. <lb/>Recruitment of polII to ACT1 was unaffected in the SCF mutants. <lb/>Results from all the ChIP analyses closely parallel the transcription <lb/>results and imply that the UPS is important for sustaining the <lb/>interaction of RNA polymerase II with the targets of some activators <lb/>despite the increased accumulation of activator (for Gcn4) at the <lb/>promoter. <lb/>Figure 2 | Defects in the UPS lead to the accumulation of ubiquitinated <lb/>Gcn4 and impair the association of RNA polymerase II with Gcn4 and Gal4 <lb/>targets. a, pdr5D strains expressing Gcn4–Myc9 (WT) or Gcn4–3T2S– <lb/>Myc9 (3T2S) were grown in minimal medium and treated with MG132 or <lb/>DMSO. Immunoprecipitations (IPs) were performed with 9E10 antibodies <lb/>recognizing Myc9, and subsequent western blots of the input (lanes 1–4) or <lb/>immunoprecipitation (IP; lanes 5–12) samples were probed with 9E10 (lanes <lb/>1–8) or anti-Ub antibodies (lanes 9–12). b, A WT strain, as above, was <lb/>processed for ChIP analysis with 9E10 and anti-polII antibodies. PCR was <lb/>performed to amplify the promoter, ORF and terminator regions of HIS4 <lb/>and the ORF of ACT1. c, ChIP analysis of Gal4–TAP and polII was <lb/>performed with galactose-induced strains treated with MG132 or DMSO. <lb/>The promoter and ORF regions of GAL1 were amplified. IgG was used to <lb/>retrieve Gal4–TAP. d, ChIP analysis of Gcn4–Myc9 and polII was performed, <lb/>as in b, with WT, cdc34-2 and cdc4-1 strains that were grown at 27 8C, then <lb/>shifted to 30 8C for 1 h. kb, kilobases; n.d., not done. <lb/></body> <note place="headnote">LETTERS <lb/>NATURE|Vol 438|3 November 2005 <lb/></note> <page>114 <lb/></page> <note place="footnote">© 2005 Nature Publishing Group <lb/></note> <body>To test whether turnover of the activator itself can promote <lb/>function, we evaluated the stabilized Gcn4–3T2S (ref. 7). In minimal <lb/>medium the levels of total protein (Fig. 3a) and promoter-associated <lb/>Gcn4–Myc9 (Fig. 3b) were about twofold to threefold higher in the <lb/>gcn4–3T2S strain. Despite such increases, gcn4–3T2S did not alter the <lb/>expression of Gcn4 targets in minimal (Fig. 3c, SM þ ; Fig. 3e, <lb/>CDC34) or starvation medium (Fig. 3c, 2Leu), indicating a <lb/>possible decrease in specific activity. Most importantly, gcn4–3T2S <lb/>partly alleviated the deleterious effects of an impaired UPS on <lb/>Gcn4-dependent transcription. For example, MG132 inhibited <lb/>HIS4 transcription in gcn4–3T2S much less than in GCN4 (Fig. 3d). <lb/>In addition, cdc34-2 diminished HIS3P–LacZ expression only about <lb/>1.2-fold in gcn4–3T2S, in comparison with more than 2.5-fold in <lb/>GCN4 (Fig. 3e). The cdc34-2 mutation had no impact on gcn4D <lb/>(Fig. 3e). A similar epistatic relationship was seen on deletion of <lb/>SRB10 and PHO85, which stabilizes Gcn4 to a similar extent to the <lb/>gcn4–3T2S mutation 7 . Deletion of both CDKs (DD) slightly increased <lb/>LacZ expression (1.2-fold) and, as with gcn4–3T2S, cdc34-2 only <lb/>mildly affected expression (1.2-fold) in the DD strain (Fig. 3f). The <lb/>suppression of cdc34-2 required the deletion of both kinases, because <lb/>srb10D and pho85D single mutants remained relatively sensitive to <lb/>cdc34-2 (Fig. 3f). We note that gcn4–3T2S is not completely refrac-<lb/>tory to UPS inhibition, indicating that, in addition to Gcn4, the UPS <lb/>might also promote transcription through other factors. Neverthe-<lb/>less, these findings indicate that proteolysis of CDK-phosphorylated <lb/>Gcn4 by means of the UPS might be important in sustaining <lb/>maximal expression of Gcn4 targets and that, in the absence of <lb/>phosphorylation, Gcn4 activity is less dependent on its turnover. <lb/>Components of the UPS have been posited to activate transcrip-<lb/>tion by multiple mechanisms 2,3 . In numerous examples, including <lb/>the activation of Gcn4 in response to ultraviolet radiation 19 and <lb/>transcription of NF-kB (ref. 2) and oestrogen receptor targets 20 , the <lb/>UPS seems to mediate signalling upstream of the activator. As <lb/>discussed in Supplementary Fig. S3, this mechanism probably does <lb/>not account for our findings. Meanwhile, subunits of the 19S cap of <lb/>the proteasome have been suggested to have a positive role in <lb/>transcription that is independent of their proteolytic function 21,22 . <lb/>In addition, it has been proposed that the ubiquitination of <lb/>Gal4–VP16 and c-Myc transiently increases the activity of these <lb/>factors before proteolysis 15–17 . Our results differ substantively from <lb/>these examples, in that neither the 19S cap (whose activity is not <lb/>known to be affected by inhibition of the 20S proteases 23 ) nor <lb/>ubiquitination was sufficient to achieve maximal transcription of <lb/>Gcn4 targets (Figs 1d and 2a). Instead, we found that inhibition of <lb/>the proteasome and genetic manipulations of the UPS, the CDKs for <lb/>Gcn4, and Gcn4 itself all provided evidence that turnover of Gcn4 <lb/>normally enhances its function. <lb/>Proteasome activity also seems to sustain inducible transcription <lb/>mediated by Gal4 and Ino2/4. Interestingly, activation of promoter-<lb/>associated Gal4 in galactose medium requires Srb10-dependent <lb/>phosphorylation 24,25 , and this activated form has a short half-life 24 . <lb/>This indicates that Gal4 might be regulated by degradation in a <lb/>manner similar to that of Gcn4. We previously proposed a model <lb/>consistent with these current findings in which proteolysis is required <lb/>to remove 'spent' activators and to reset the promoter 3 . The initial <lb/>'pioneer round(s)' of transcription would not involve the UPS, but <lb/>subsequent rounds would be stimulated by turnover of the spent, <lb/>promoter-bound activator to allow binding of a fresh molecule. This <lb/>mechanism places Gcn4, Gal4 and Ino2/4 into a class of regulatory <lb/>factors—including securin, p21 and p27—whose activity is required <lb/>early in a process but whose subsequent turnover or removal <lb/>promotes completion of the process or subsequent reaction cycles. <lb/>We call this phenomenon 'activation by destruction' and believe that, <lb/>given the diversity of the examples listed in Supplementary Table S2, <lb/>it might represent a regulatory mechanism for a large class of factors <lb/>and might be an important determinant of infection and disease. <lb/>METHODS <lb/>Yeast strains, growth conditions and extract preparation. A complete list of <lb/>yeast strains used in this study is provided in Supplementary Table S1. All strains <lb/>were derived from the S288C background, except RJD 2505 and RJD 3137–3141, <lb/>which were derived from the W303 background. Strains were constructed and <lb/>grown in accordance with standard protocols 26 . A description of the various <lb/>media used in the study is given in Supplementary Information. MG132 <lb/>(American Peptide) was added to cultures of pdr5D strains to a final concen-<lb/>tration of 50 mM. All extracts were prepared by lysis with glass beads (Sigma) in a <lb/>Fast Prep (Bio 101) device. Ubiquitin derivative analysis (Fig. 1d) is described in <lb/>the Supplementary Methods. <lb/>RT–PCR analysis. mRNA was prepared using RNeasy kits (Qiagen). Total <lb/>mRNA (200 ng) and 10 pmol of oligo(dT) were used to reverse-transcribe <lb/>complementary DNA (Stratagene). One-tenth of the cDNA reaction was then <lb/>used for 20–22 cycles of PCR and products were resolved on 2% agarose gels. <lb/>Primer sequences are available from the authors on request. <lb/>b-Galactosidase assays. The HIS3P–LacZ, HIS4P–LacZ and GCRE–LacZ repor-<lb/>ter constructs and the protocol to measure b-galactosidase activity were as <lb/>described previously 27 . Reported activity was normalized to total extract protein <lb/>as measured by bicinchoninic acid assay (Pierce). Relative activities are reported <lb/>with average WT activity set to 1. <lb/>Western blots. Except as noted, extracts were prepared by immediate boiling of <lb/>cell pellets in 2 £ Laemmli SDS sample buffer followed by lysis with glass beads. <lb/>Equal amounts of total protein (as judged by Coomassie staining) were resolved <lb/>by SDS–PAGE. Blots were probed with 9E10 antibodies to recognize Gcn4–Myc9 <lb/>or FK1 antibodies (Affiniti) to recognize ubiquitin. Horseradish peroxidase-<lb/>coupled goat anti-mouse secondary antibodies (Bio-Rad) were used for <lb/>detection. <lb/>Figure 3 | The UPS has little effect on the activity of stable, <lb/>non-phosphorylated versions of Gcn4. a, GCN4 (WT), gcn4–3T2S or <lb/>gcn4D strains were grown in minimal medium and processed for western <lb/>blotting with 9E10 antibodies. Protein half-life data (t 1/2 ) were reported <lb/>previously 7 . n.a., not applicable. b, ChIP analysis of the above strains was <lb/>performed for the HIS4 promoter. c, RT–PCR of the indicated transcripts <lb/>were performed with WTand gcn4–3T2S strains grown in minimal (Min) or <lb/>starvation (2Leu) medium. d, RT–PCR analysis of HIS4 and ACT1 was <lb/>performed with WTand 3T2S strains in the presence and absence of MG132. <lb/>e, CDC34 (open bars) and cdc34-2 (filled bars) strains with the HIS3P-LacZ <lb/>reporter and expressing either WT, 3T2S or null (gcn4D) versions of <lb/>GCN4 were grown in minimal medium and processed for b-galactosidase <lb/>(b-gal) activity. Standard deviations were calculated from three replicates. <lb/>f, WTand cdc34-2 strains with a HIS4P-LacZ reporter and harbouring WTor <lb/>deleted versions of SRB10 and/or PHO85 were treated as in e. <lb/></body> <note place="headnote">NATURE|Vol 438|3 November 2005 <lb/>LETTERS <lb/></note> <page>115 <lb/></page> <note place="footnote">© 2005 Nature Publishing Group <lb/></note> <body>Immunoprecipitations and detection of ubiquitinated Gcn4. DMSO-treated <lb/>or MG132-treated cultures were treated with formaldehyde (final concentration <lb/>1%) for 20 min to trap ubiquitinated intermediates. Crosslinking was quenched <lb/>and extracts were made in ChIP buffer 18 . A 10% sample of the extract (whole cell <lb/>extract) was removed and boiled in SDS sample buffer. The remainder of <lb/>the extract was incubated at 4 8C with 9E10 antibodies coupled to Protein A– <lb/>Sepharose (Sigma) beads. Proteins were eluted and crosslinks were reversed by <lb/>being boiled in SDS sample buffer. Proteins samples were then processed for <lb/>western blotting. For FK1 (anti-ubiquitin) western blots, the nitrocellulose was <lb/>boiled before incubation with the antibody. <lb/>ChIP assays. ChIP assays were performed as described 18 ; 9E10 antibodies were <lb/>used to immunoprecipitate chromatin fragments associated with Gcn4–Myc9 and <lb/>antibodies against the carboxy-terminal domain of the largest subunit of RNA <lb/>polymerase II (anti-polII; Covance) were used to immunoprecipitate fragments <lb/>associated with polII. Protein G–Sepharose beads (Amersham Biosciences) <lb/>were used to precipitate antibody–antigen complexes. Rabbit immunoglobulin <lb/>(Ig) G–agarose (Sigma) was used to immunoprecipitate Gal4-associated <lb/>fragments. Primer sequences are available from the authors on request. <lb/></body> <front>Received 2 June; accepted 3 August 2005. <lb/></front> <listBibl>1. Pickart, C. M. Back to the future with ubiquitin. Cell 116, 181–-190 (2004). <lb/>2. Muratani, M. & Tansey, W. P. How the ubiquitin–-proteasome system controls <lb/>transcription. Nature Rev. Mol. Cell Biol. 4, 192–-201 (2003). <lb/>3. Lipford, J. R. & Deshaies, R. J. Diverse roles for ubiquitin-dependent proteolysis <lb/>in transcriptional activation. Nature Cell Biol. 5, 845–-850 (2003). <lb/>4. Lee, D. H. & Goldberg, A. L. Proteasome inhibitors: valuable new tools for cell <lb/>biologists. Trends Cell Biol. 8, 397–-403 (1998). <lb/>5. Meimoun, A. et al. Degradation of the transcription factor Gcn4 requires the <lb/>kinase Pho85 and the SCF(CDC4) ubiquitin-ligase complex. Mol. Biol. Cell 11, <lb/>915–-927 (2000). <lb/>6. Finley, D. et al. Inhibition of proteolysis and cell-cycle progression in a <lb/>multiubiquitination-deficient yeast mutant. Mol. Cell. Biol. 14, 5501–-5509 <lb/>(1994). <lb/>7. Chi, Y. et al. Negative regulation of Gcn4 and Msn2 transcription factors by <lb/>Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078–-1092 (2001). <lb/>8. Adams, J. & Kauffman, M. Development of the proteasome inhibitor Velcade <lb/>(bortezomib). Cancer Invest. 22, 304–-311 (2004). <lb/>9. Mitsiades, N. et al. Molecular sequelae of proteasome inhibition in human <lb/>multiple myeloma cells. Proc. Natl Acad. Sci. USA 99, 14374–-14379 (2002). <lb/>10. Fleming, J. A. et al. Complementary whole-genome technologies reveal the <lb/>cellular response to proteasome inhibition by PS-341. Proc. Natl Acad. Sci. USA <lb/>99, 1461–-1466 (2002). <lb/>11. Hinnebusch, A. G. & Natarajan, K. Gcn4p, a master regulator of gene <lb/>expression, is controlled at multiple levels by diverse signals of starvation and <lb/>stress. Eukaryot. Cell 1, 22–-32 (2002). <lb/>12. Kornitzer, D., Raboy, B., Kulka, R. G. & Fink, G. R. Regulated degradation of the <lb/>transcription factor GCN4. EMBO J. 13, 6021–-6030 (1994). <lb/>13. Albrecht, G., Mosch, H. U., Hoffmann, B., Reusser, U. & Braus, G. H. Monitoring <lb/>the Gcn4 protein-mediated response in the yeast Saccharomyces cerevisiae. <lb/>J. Biol. Chem. 273, 12696–-12702 (1998). <lb/>14. Hilt, W., Enenkel, C., Gruhler, A., Singer, T. & Wolf, D. H. The PRE4 gene codes <lb/>for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-<lb/>hydrolyzing activity. Mutations link the proteasome to stress-and ubiquitin-<lb/>dependent proteolysis. J. Biol. Chem. 268, 3479–-3486 (1993). <lb/>15. Salghetti, S. E., Caudy, A. A., Chenoweth, J. G. & Tansey, W. P. Regulation of <lb/>transcriptional activation domain function by ubiquitin. Science 293, 1651–-1653 <lb/>(2001). <lb/>16. Kim, S., Herbst, A., Tworkowski, K., Salghetti, S. & Tansey, W. Skp2 regulates <lb/>Myc protein stability and activity. Mol. Cell 11, 1177–-1188 (2003). <lb/>17. von der Lehr, N. et al. The F-Box protein Skp2 participates in c-Myc <lb/>proteosomal degradation and acts as a cofactor for c-Myc-regulated <lb/>transcription. Mol. Cell 11, 1177–-1188 (2003). <lb/>18. Aparicio, O., Geisberg, J. & Struhl, K. in Current Protocols in Molecular Biology <lb/>(eds Ausubel, F. M. et al.) 21.3.1–-21.3.12 (Wiley, New York, 2004). <lb/>19. Stitzel, M. L., Durso, R. & Reese, J. C. The proteasome regulates the <lb/>UV-induced activation of the AP-1-like transcription factor Gcn4. Genes Dev. <lb/>15, 128–-133 (2001). <lb/>20. Reid, G. et al. Cyclic, proteasome-mediated turnover of unliganded and <lb/>liganded ERa on responsive promoters is an integral feature of estrogen <lb/>signalling. Mol. Cell 11, 695–-707 (2003). <lb/>21. Gonzalez, F., Delahodde, A., Kodadek, T. & Johnston, S. A. Recruitment of a 19S <lb/>proteasome subcomplex to an activated promoter. Science 296, 548–-550 <lb/>(2002). <lb/>22. Morris, M. C. et al. Cks1-dependent proteasome recruitment and activation of <lb/>CDC20 transcription in budding yeast. Nature 423, 1009–-1013 (2003). <lb/>23. Verma, R. et al. Deubiquitination and degradation of proteins by the 26S <lb/>proteasome requires the Rpn11 metalloprotease motif. Science 298, 611–-615 <lb/>(2002). <lb/>24. Muratani, M., Kung, C., Shokat, K. M. & Tansey, W. P. The F box protein <lb/>Dsg1/Mdm30 is a transcriptional coactivator that stimulates Gal4 turnover <lb/>and cotranscriptional mRNA processing. Cell 120, 887–-899 (2005). <lb/>25. Hirst, M., Kobor, M. S., Kuriakose, N., Greenblatt, J. & Sadowski, I. GAL4 is <lb/>regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent <lb/>protein kinase SRB10/CDK8. Mol. Cell 3, 673–-678 (1999). <lb/>26. Guthrie, C. & Fink, G. R. Guide to Yeast Genetics and Molecular Biology <lb/>(Academic, San Diego, 1991). <lb/>27. Drysdale, C. M. et al. The transcriptional activator GCN4 contains multiple <lb/>activation domains that are critically dependent on hydrophobic amino acids. <lb/>Mol. Cell. Biol. 15, 1220–-1233 (1995). <lb/></listBibl> <div type="annex" subtype="supplementary information">Supplementary Information is linked to the online version of the paper at <lb/>www.nature.com/nature.<lb/></div> <div type="acknowledgement">Acknowledgements We thank D. Finley, D. H. Wolf, R. Young, A. Hinnebusch, <lb/>J. Shaw, B. Westermann, J. Nunnari and G. Braus for gifts of strains and <lb/>reagents; B. Tansey for communicating results before publication; and J. Shaw, <lb/>B. Westermann, J. Nunnari, S. Sadis, A. Ansari and the members of the Deshaies <lb/>laboratory for comments and criticism. This work was supported in part by an <lb/>NIH Research Project Grant to R.J.D. R.J.D. is an Investigator of the Howard <lb/>Hughes Medical Institute. J.R.L. was supported by an NIH National Research <lb/>Service Award and a Caltech-Amgen Postdoctoral Fellowship. <lb/></div> <div type="annex">Author Information Reprints and permissions information is available at <lb/>npg.nature.com/reprintsandpermissions. The authors declare no competing <lb/>financial interests. Correspondence and requests for materials should be <lb/>addressed to R.J.D. (deshaies@caltech.edu). <lb/></div> <note place="headnote">LETTERS <lb/>NATURE|Vol 438|3 November 2005 <lb/></note> <page>116 </page> </text> </tei>