Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.

Mediator is an evolutionarily conserved complex best known for its role as a coactivator responsible for transducing regulatory signals from DNA-bound activators to the basal RNA polymerase II (Pol II) machinery that initiates transcription from promoters of protein-encoding genes. By exploiting our in vivo activator-independent transcription assay in Saccharomyces cerevisiae, in combination with new temperature sensitive (ts) mutants of Med14 N-terminal half exhibiting widespread transcriptional defects, and existing ts mutants of Kin28 and Med17, we show that, in the absence of activator: (i) Mediator can associate with a promoter as a form devoid of the Cyclin-dependent kinase 8 (CDK8) module, and this association remains regulated by Kin28; (ii) Mediator can stimulate the assembly of the entire Pol II initiation machinery. Although the literature emphasizes the role of the interaction between activators and Mediator, together our results support the view that Mediator is able to act through a dual mechanism in vivo, activator-dependent but also activator-independent, therefore not always as a coactivator.

Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1–6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.

Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.

Redox homeostasis is achieved by antioxidant systems, involving a large collection of enzymes that scavenge or degrade Reactive Oxygen Species (ROS) produced endogenously at low levels during cell growth. Besides the enzymatic protection against ROS, cells also contain small antioxidant molecules, such as glutathione (GSH). With an intracellular concentration between 1 and 10 mM, GSH is the most abundant non-protein thiol in the cell and is considered as the major redox buffer of the cell. In a previous study, we showed that only scarce amounts of intracellular GSH are required to protect yeast nuclear activities during oxidative stress. Surprisingly, such protection is sufficient for cell survival despite the strong oxidation of cytosolic proteins and the complete inhibition of protein synthesis (Hatem et al., 2014). GSH synthesis is a two-step process involving the gamma-glutamylcysteine (g-GC) synthetase Gsh1, which produces the g-GC intermediate from glutamate and cysteine, and the glutathione synthetase Gsh2, which adds a glycine to g-GC to release the final tripeptide. Deletion of GSH2 leads to yeast cells accumulating abnormal amounts of g-GC. It has been suggested that this molecule could replace GSH during oxidative stress exposure as the viability of ∆gsh2 cells, unable to synthesize GSH, is only mildly affected in oxidative stress conditions. Because our previous study revealed that the antioxidant protection of all cellular components and activities is not strictly required to preserve cell viability during oxidative stress, we decided to better characterize the physiological response of ∆gsh2 cells submitted to hydrogen peroxide treatments. Here we present the main results of this study and discuss the potential role of g-GC in the cellular protection against oxidative injury, compare to GSH.

Mediator is a prominent multisubunit coactivator that functions as a bridge between gene-specific activators and the basal RNA polymerase (Pol) II initiation machinery. Here, we study the poorly documented role of Mediator in basal, or activator-independent, transcription in vivo. We show that Mediator is still present at the promoter when the Pol II machinery is recruited in the absence of an activator, in this case through a direct fusion between a basal transcription factor and a heterologous DNA binding protein bound to the promoter. Moreover, transcription resulting from activator-independent recruitment of the Pol II machinery is impaired by inactivation of the essential Mediator subunit Med17 due to the loss of Pol II from the promoter. Our results strongly support that Mediator is an integral component of the minimal machinery essential in vivo for stable Pol II association with the promoter.

Cell adaptation to the environment often involves induction of complex gene expression programs under the control of specific transcriptional activators. For instance, in response to cadmium, budding yeast induces transcription of the sulfur amino acid biosynthetic genes through the basic-leucine zipper activator Met4, and also launches a program of substitution of abundant glycolytic enzymes by isozymes with a lower content in sulfur. We demonstrate here that transcriptional induction of PDC6, which encodes a pyruvate decarboxylase isoform with low sulfur content, is directly controlled by Met4 and its DNA-binding cofactors the basic-helix-loop-helix protein Cbf1 and the two homologous zinc finger proteins Met31 and Met32. Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes. Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors. We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.

Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification.

Chromatin immunoprecipitation (ChIP) is one of the most powerful methods to identify and characterize the association of proteins with specific genomic regions in the context of intact cells. In this method, cells are first treated with formaldehyde to crosslink protein-protein and protein-DNA complexes in situ. Next, the crosslinked chromatin is sheared by sonication to generate small chromatin fragments, and the fragments associated with the protein of interest are immunoprecipitated using antibodies to the protein. Finally, protein-DNA crosslinks are reversed and the DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (PCR). Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo. The ChIP method described here is intended for studying protein-DNA association in the budding yeast Saccharomyces cerevisiae, but it can be easily implemented in other cell types, including fly, mammalian, and plant cells.

Promoter-specific initiation of transcription by RNA polymerase II (Pol II) requires both gene-specific regulators and general transcription factors (GTFs: TFIIB, -D, -E, -F, and -H) (Woychik and Hampsey 2002Woychik N.A. Hampsey M. Cell. 2002; 108: 453-463Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Biochemical and genetic studies in yeast led to the discovery of a Mediator (MED) complex of 20 protein subunits, linking transcriptional regulators to Pol II and GTFs (Flanagan et al. 1991Flanagan P.M. Kelleher 3rd, R.J. Sayre M.H. Tschochner H. Kornberg R.D. Nature. 1991; 350: 436-438Crossref PubMed Scopus (253) Google Scholar, Kelleher et al. 1990Kelleher 3rd, R.J. Flanagan P.M. Kornberg R.D. Cell. 1990; 61: 1209-1215Abstract Full Text PDF PubMed Scopus (283) Google Scholar, Kim et al. 1994Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (872) Google Scholar). 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Biol. Chem. 2003; 278 (a): 49671-49674Crossref PubMed Scopus (43) Google Scholar, Tomomori-Sato et al. 2004Tomomori-Sato C. Sato S. Parmely T.J. Banks C.A. Sorokina I. Florens L. Zybailov B. Washburn M.P. Brower C.S. Conaway R.C. et al.J. Biol. Chem. 2004; 279: 5846-5851Crossref PubMed Scopus (22) Google Scholar). Initial studies identified 8 MEDs conserved from fungi to humans: Med6/Pmc5/ARC/DRIP33/TRAP32, Med7/ARC/DRIP/TRAP34/CRSP33, Nut2/Med10/TRAP15, Srb7/SURB7/TRAP19, Rgr1/Pmc1/ARC/CRSP/DRIP150/TRAP170, Soh1/TRAP18 (note that Soh1 has not been yet identified in purified yeast Mediator), Srb10/Ssn3/Ume5/Gig2/Nut7/Rye5/CDK8, and Srb11/Ssn8/Ume3/Gig3/Nut9/Rye2/Cyclin C) (for reviews see Malik and Roeder 2000Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, Rachez and Freedman 2001Rachez C. Freedman L.P. Curr. Opin. Cell Biol. 2001; 13: 274-280Crossref PubMed Scopus (225) Google Scholar). 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Chem. 2003; 278 (b): 15123-15127Crossref PubMed Scopus (44) Google Scholar, Spahr et al. 2001Spahr H. Samuelsen C.O. Baraznenok V. Ernest I. Huylebroeck D. Remacle J.E. Samuelsson T. Kieselbach T. Holmberg S. Gustafsson C.M. Proc. Natl. Acad. Sci. USA. 2001; 98: 11985-11990Crossref PubMed Scopus (33) Google Scholar, Tomomori-Sato et al. 2004Tomomori-Sato C. Sato S. Parmely T.J. Banks C.A. Sorokina I. Florens L. Zybailov B. Washburn M.P. Brower C.S. Conaway R.C. et al.J. Biol. Chem. 2004; 279: 5846-5851Crossref PubMed Scopus (22) Google Scholar). Further bioinformatics analyses and functional studies have revealed that the human MEDs ARC105 and yeast Gal11 harbor an activator-targeted domain related to the KIX domain found in the CBP/p300 co-activators, suggesting that ARC105 and Gal11 are evolutionarily related (Novatchkova and Eisenhaber 2004Novatchkova M. Eisenhaber F. Curr. Biol. 2004; 14: R54-R55Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; A.M.N., unpublished data). The time now seems right to establish a unified MED nomenclature in order to enhance understanding of the scientific literature by a wide audience and to aid cross-species comparisons and proper annotation of sequence databases.Table 1New Nomenclature for MED Subunits Including the Corresponding Known or Predicted Orthologs and ParalogsC. elegansH. sapiensdAcronyms given to MEDs identified from various mammalian MED-like complexes (Malik and Roeder, 2000). Many of the components listed under Others recently have been found in both the larger and smaller complexes; however, the MED12, MED13, CDK8, and CycC components clearly are not present in the smaller complexes, consistent with their absence in a subpopulation of yeast Mediator complexes.New nameS. cerevisiaeaFrom SGD.S. pombePrevious namebFrom WormBase.New nameD. melanogastercFrom FlyBase.TRAP/SMCCARC/DRIPCRSPPC2OTHERSMED1Med1Pmc2SOP-3*MDT-1.1Trap220*TRAP220ARC/DRIP205CRSP200TRAP220PBPMED1LT23C6.1*MDT-1.2MED2Med2MED3Pgd1/Hrs1/Med3MED4Med4Pmc4/SpMed4ZK546.13*MDT-4Trap36TRAP36ARC/DRIP36TRAP36p34MED5Nut1MED6Med6Pmc5/SpMed6LET-425/MED-6MDT-6Med6hMed6ARC/DRIP33hMed6p32MED7Med7SpMed7LET-49/MED-7MDT-7Med7*hMed7ARC/DRIP34CRSP33hMed7p36MED8Med8Sep15/SpMed8Y62F5A.1b*MDT-8Arc32*ARC32mMed8MED9Cse2/Med9CG5134*Med25MED10Nut2/Med10SpNut2T09A5.6MDT-10Nut2*hNut2hMed10hNut2MED11Med11R144.9*MDT-11Med21HSPC296MED12Srb8SpSrb8DPY-22/SOP-1*MDT-12Kto*TRAP230ARC/DRIP240MED12LTRALPUSH*MED13Ssn2/Srb9SpTrap240LET-19*MDT-13Skd/Pap/Bli*TRAP240ARC/DRIP250MED13LPROSIT240MED14Rgr1Pmc1/SpRgr1RGR-1*MDT-14Trap170TRAP170ARC/DRIP150CRSP150TRAP170p110MED15Gal11SpGal11*R12B2.5b*MDT-15Arc105*ARC105PCQAPTIG-1MED16Sin4Trap95*TRAP95DRIP92TRAP95p96bMED17Srb4SpSrb4Y113G7B.18*MDT-17Trap80TRAP80ARC/DRIP77CRSP77TRAP80p78MED18Srb5Pmc6/Sep11C55B7.9*MDT-18p28/CG14802p28bMED19Rox3SpRox3Y71H2B.6*MDT-19CG5546*LCMR1MED20Srb2SPAC17G8.05*Y104H12D.1*MDT-20TrfphTRFPhTRFPp28aMED21Srb7SpSrb7C24H11.9*MDT-21Trap19hSrb7hSrb7hSrb7p21MED22Srb6SpSrb6ZK970.3*MDT-22Med24Surf5MED23SUR-2*MDT-23Trap150β*TRAP150βARC/DRIP130CRSP130TRAP150βhSur2MED24Trap100*TRAP100ARC/DRIP100CRSP100TRAP100MED25Arc92*ARC92ACID1MED26Arc70*ARC70CRSP70MED27Pmc3T18H9.6*MDT-27Trap37*TRAP37CRSP34TRAP37MED28W01A8.1*MDT-28Med23Fksg20MED29K08E3.8*MDT-29Intersex*HintersexMED30Trap25TRAP25MED31Soh1*SpSoh1/Sep10*F32H2.2*MDT-31Trap18hSoh1hSoh1CDK8Srb10/Ssn3/Ume5SpSrb10CDK-8*Cdk8hSrb10CDK8CycCSrb11/Ssn8/Ume3SpSrb11H14E04.5*CIC-1CycChSrb11CycCAsterisks indicate that the corresponding proteins have not yet been identified in purified MED complexes.a From SGD.b From WormBase.c From FlyBase.d Acronyms given to MEDs identified from various mammalian MED-like complexes Malik and Roeder 2000Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar. Many of the components listed under Others recently have been found in both the larger and smaller complexes; however, the MED12, MED13, CDK8, and CycC components clearly are not present in the smaller complexes, consistent with their absence in a subpopulation of yeast Mediator complexes. Open table in a new tab Asterisks indicate that the corresponding proteins have not yet been identified in purified MED complexes. The unified nomenclature, shown in Table 1, is based on the following considerations:1.The new nomenclature complies with guidelines endorsed by the Saccharomyces Genome Database (SGD), the FlyBase and WormBase resources, and the human HUGO Gene Nomenclature Committees.2.MED is the most explicit acronym.3.This nomenclature acknowledges the discovery of MED complexes in yeast.4.In light of point 3, the original yeast MEDs will retain their names (MED1-11; note that the MED5 acronym will replace Nut1).5.The remaining yeast MEDs will be given names starting from MED12, in order of decreasing conceptual molecular weights deduced from primary sequences.6.MEDs found outside budding yeast will be given names starting from MED23 in order of decreasing calculated molecular weights (based on the human protein). At present, this list extends to MED31.7.Future bona fide new MED components will be assigned numbers starting from MED32.8.The general nomenclature will employ CDK8 and CycC, as the CDK-cyclin couple is readily identifiable for a wide scientific audience.9.Except for the specific case of C. elegans (see point 10), paralogs in the same organism will be termed MED-like, e.g., MED12L in humans.10.C. elegans MEDs will retain the specific nomenclature already adopted by WormBase, the MED acronym being used for another gene category. Thus MDT-6 (for eiaor-6) replaces MED6, but the proposed numbering from 1 to 31 would be retained. In addition, following usual recommendations in this organism, the two MED1 paralogs would be called MDT-1.1 and MDT-1.2. We believe the relative simplicity of the new, common nomenclature will expedite functional comparisons in different species, while remaining flexible enough to accommodate additional species-specific MEDs as they arise. Some uncertainties persist concerning the assignments of orthologous subunits, and the nomenclature can be updated if new data so require. To facilitate communication between researchers working inside and outside of the transcription field, we recommend that this numbering system be used in all future publications concerning Mediator complexes.

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro . Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo . We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Mot1 stably associates with the TATA-binding protein (TBP), and it can dissociate TBP from DNA in an ATP-dependent manner. Mot1 acts as a negative regulator of TBP function in vitro, but genome-wide transcriptional profiling suggests that Mot1 positively affects about 10% of yeast genes and negatively affects about 5%. Unexpectedly, Mot1 associates with active RNA polymerase (Pol) II and III promoters, and it is rapidly recruited in response to activator proteins. At Pol II promoters, Mot1 association requires TBP and is strongly correlated with the level of TBP occupancy. However, the Mot1/TBP occupancy ratio at both Mot1-stimulated and Mot1-inhibited promoters is high relative to that at typical promoters, strongly suggesting that Mot1 directly affects transcriptional activity in a positive or negative manner, depending on the gene. The effect of Mot1 at the HIS3 promoter region depends on the functional quality and DNA sequence of the TATA element. Unlike TBP, Mot1 association is largely independent of the Srb4 component of Pol II holoenzyme, and it also can occur downstream of the promoter region. Mot1 removes TBP, but not TBP complexes or preinitiation complexes, from inappropriate genomic locations. Mot1 inhibits the association of NC2 with promoters, suggesting that the TBP-Mot1 and TBP-NC2 complexes compete for promoter occupancy in vivo. We speculate that Mot1 does not form transcriptionally active TBP complexes but rather regulates transcription in vivo by modulating the activity of free TBP and/or by affecting promoter DNA structure.

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 SCF(Met30) 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.

In yeast, TFIID strongly associates with nearly all ribosomal protein (RP) promoters, but a TAF-independent form of TBP preferentially associates with other active promoters. RP promoters are regulated in response to growth stimuli, in most cases by a Rap1-containing activator. This Rap1-dependent activator is necessary and sufficient for TFIID recruitment, whereas other activators do not efficiently recruit TFIID. TAFs are recruited to RP promoters even when TBP and other general transcription factors are not associated, suggesting that TFIID recruitment involves a direct activator-TAF interaction. Most RP promoters lack canonical TATA elements, and they are preferentially activated by the Rap1-containing activator. These results demonstrate activator-specific recruitment of TFIID in vivo, and they suggest that TFIID recruitment is important for coordinate expression of RP genes.

Transcriptional activity in yeast strongly correlates with promoter occupancy by general factors such as TATA binding protein (TBP), TFIIA, and TFIIB, but not with occupancy by TBP-associated factors (TAFs). Thus, TBP exists in at least two transcriptionally active forms in vivo. The TAF-containing form corresponds to the TFIID complex, whereas the form lacking TAFs corresponds to TBP itself or to some other TBP complex. Heat shock treatment altered the relative utilization of these TBP forms, with TFIID being favored. Promoter-specific variations in the association of these distinct forms of TBP may explain why only some yeast genes require TFIID for transcriptional activity in vivo.

In eukaryotes, transcriptional activators have been proposed to function by recruiting the RNA polymerase II (Pol II) machinery1,2,3, by altering the conformation of this machinery4,5, or by affecting steps after initiation6,7,8, but the evidence is not definitive. Genomic footprinting of yeast TATA-box elements reveals activator-dependent alterations of chromatin structure9 and activator-independent protection10, but little is known about the association of specific components of the Pol II machinery with promoters in vivo. Here we analyse TATA-box-binding-protein (TBP) occupancy of 30 yeast promoters in vivo. We find that TBP association with promoters is stimulated by activators and inhibited by the Cyc8–Tup1 repressor, and that transcriptional activity correlates strongly with the degree of TBP occupancy. In a small subset of promoters, TBP occupancy is higher than expected when gene activity is low, and the activator-dependent increase is modest. TBP association depends on the PolII holoenzyme component Srb4, but not on the Kin28 subunit of the transcription factor TFIIH, even though both proteins aregenerally required for transcription. Thus in yeast cells, TBP association with promoters occurs in concert with the Pol II holoenzyme, activator-dependent recruitment of the Pol II machinery occurs at the vast majority of promoters, and Kin28 acts after the initial recruitment.

We have made a DNA microarray that includes not only all the open reading frames (ORFs) and other features in the yeast genome, but also all the intergenic regions. We are using this as a tool to construct genome-wide maps of DNA-protein interactions for proteins that interact directly or indirectly with DNA or chromatin in vivo. Proteins are crosslinked to DNA in vivo using formaldehyde and the crosslinked DNA is extracted and sheared. DNA specifically associated with any protein of interest is immunoprecipitated using a specific antibody against the protein or an epitope tag that may be fused to the protein. The selected DNA, representing loci that the protein interacts with in vivo, can be identified by fluorescently labelling it and hybridizing it to the microarray along with an appropriate reference probe. This approach is being used to map the genome-wide interactions of sequence-specific DNA binding proteins, components of the transcription machinery and chromatin components, under a variety of conditions.

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCFCdc4 complex). Skp1 binds a motif called the F-box and in turn F-box proteins appear to recruit specific substrates for ubiquitination. We find that Skp1 interacts with Cdc53 in vivo, and that Skp1 bridges Cdc53 to three different F-box proteins, Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1 suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1, Grr1 is specific for degradation of the G1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes.

In eukaryotes, gene expression depends on activatorproteins that bind enhancer elements and stimulate transcription by RNA polymerase II (pol II) (Struhl 1995;Zawel and Reinberg 1995). This general requirement foractivators is inferred from numerous observations invivo that intact promoters are much more efficientlytranscribed than core promoter derivatives containingonly the TATA and initiator elements. The pol II transcription machinery is complex and has a molecularweight comparable to that of a ribosome. The pol II machinery is composed of two basic components, TFIIDand the pol II holoenzyme. The TFIID complex, whichcontains the TATA-binding protein (TBP) and TBP-associated factors (TAFs), specifically binds the core promoter region; TBP interacts with high affinity and specificity for TATA elements, whereas certain TAFs caninteract with some specificity for initiator and downstream elements (Burley and Roeder 1996; Verrijzer andTjian 1996; Burke and Kadonaga 1997). The pol IIholoenzyme contains the core subunits of the enzyme,basic transcription factors (e.g., TFIIB), as well as Srb,Med, and a variety of other proteins (Koleske and Young1995; Myers et al. 1998)...