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Factors Affecting Saccharomyces cerevisiae ADH2Chromatin Remodeling and Transcription

1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês

10.1074/jbc.272.49.30828

1083-351X

Loredana Verdone, Francesca Cesari, Clyde L. Denis, Ernesto Di Mauro, Micaela Caserta,

Genomics and Chromatin Dynamics

The chromatin structure of theSaccharomyces cerevisiae ADH2 gene is modified during the switch from repressing (high glucose) to derepressing (low glucose) conditions of growth. Loss of protection toward micrococcal nuclease cleavage for the nucleosomes covering the TATA box and the RNA initiation sites (−1 and +1, respectively) is the major modification taking place and is strictly dependent on the presence of the transcriptional activator ADR1.To identify separate functions involved in the transition from a repressed to a transcribing promoter, we have analyzed theADH2 chromatin organization in various genetic backgrounds. Deletion of the CCR4 gene coding for a general transcription factor impaired ADH2 expression without affecting chromatin remodeling. Growing yeast at 37 °C also resulted in chromatin remodeling at the ADH2 locus even under glucose repressing conditions. However, although this temperature-induced remodeling was dependent on the ADR1 protein, noADH2 mRNA was observed. In addition, inactivating RNA polymerase II (and therefore, elongation) was found to have no effect on the ability to reconfigure nucleosomes. Taken together, these data indicate that chromatin remodeling by itself is insufficient to induce transcription at the ADH2 promoter. The chromatin structure of theSaccharomyces cerevisiae ADH2 gene is modified during the switch from repressing (high glucose) to derepressing (low glucose) conditions of growth. Loss of protection toward micrococcal nuclease cleavage for the nucleosomes covering the TATA box and the RNA initiation sites (−1 and +1, respectively) is the major modification taking place and is strictly dependent on the presence of the transcriptional activator ADR1. To identify separate functions involved in the transition from a repressed to a transcribing promoter, we have analyzed theADH2 chromatin organization in various genetic backgrounds. Deletion of the CCR4 gene coding for a general transcription factor impaired ADH2 expression without affecting chromatin remodeling. Growing yeast at 37 °C also resulted in chromatin remodeling at the ADH2 locus even under glucose repressing conditions. However, although this temperature-induced remodeling was dependent on the ADR1 protein, noADH2 mRNA was observed. In addition, inactivating RNA polymerase II (and therefore, elongation) was found to have no effect on the ability to reconfigure nucleosomes. Taken together, these data indicate that chromatin remodeling by itself is insufficient to induce transcription at the ADH2 promoter. Transcriptional regulation has been extensively studied both by genetic and biochemical approaches. The overall complexity of the eukaryotic genome and the large number of genes to be regulated are such that repression and chromosome compaction are necessarily the dominant feature. According to this concept, the wrapping of DNA around nucleosomes is considered the easiest way to keep the majority of the genome silent. Activation would then be achieved by different combinations of positive factors. During the last 10 years, a more complex picture relating chromatin to gene activation has emerged (1Wallrath L.L. Lu Q. Granok H. Elgin S.C. Bioessays. 1994; 16: 165-170Crossref PubMed Scopus (148) Google Scholar, 2Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 3Struhl K. Cell. 1996; 84: 179-182Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 4Wolffe A.P. Pruss D. Curr. Biol. 1996; 6: 234-237Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Extensive work by Grunstein and co-workers (Refs. 5Fisher-Adams G. Grunstein M. EMBO J. 1995; 14: 1468-1477Crossref PubMed Scopus (65) Google Scholar and 6Lenfant F. Mann R.K. Thomsen B. Ling X. Grunstein M. EMBO J. 1996; 15: 3974-3985Crossref PubMed Scopus (74) Google Scholar and references therein) has pointed out that each specific histone is involved in the activation or repression of defined genes and that different histone domains have different regulatory functions. As a consequence, nucleosomes would hold more important functions than just folding and wrapping the huge mass of eukaryotic DNA. A direct involvement of specific core particles in gene activation is suggested by the finding that multiple factors acting on a promoter interact successfully with each other due to the presence of a precisely positioned nucleosome (7Schild C. Claret F.-X. Wahli W. Wolffe A.P. EMBO J. 1993; 12: 423-433Crossref PubMed Scopus (178) Google Scholar, 8Truss M. Bartsch J. Schelbert A. Haché R.J.G. Beato M. EMBO J. 1995; 14: 1737-1751Crossref PubMed Scopus (265) Google Scholar). In this respect, the in vivostudy of the exact location of nucleosomes (9Buttinelli M. Camilloni G. Costanzo G. Negri R. Venditti P. Venditti S. Di Mauro E. Methods Mol. Genet. 1995; 6: 168-185Crossref Scopus (8) Google Scholar, 10Fragoso G. John S. Roberts M.S. Hager G. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 11Tanaka S. Livingstone-Zatchej M. Thoma F. J. Mol. Biol. 1996; 257: 919-934Crossref PubMed Scopus (60) Google Scholar) as well as thein vitro analysis of the rules governing their positioning (12Linxweiler W. Hözz W. Cell. 1985; 42: 281-290Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 13Shrader T.E. Crothers D.M. J. Mol. Biol. 1990; 216: 69-84Crossref PubMed Scopus (171) Google Scholar, 14Buttinelli M. Negri R. Di Marcotullio L. Di Mauro E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10747-10751Crossref PubMed Scopus (10) Google Scholar, 15Caserta M. Di Mauro E. Bioessays. 1996; 18: 947-955Crossref Scopus (8) Google Scholar, 16Travers A.A. Muyldermans S.V. J. Mol. Biol. 1996; 257: 486-491Crossref PubMed Scopus (38) Google Scholar) become relevant for the understanding of regulatory mechanisms. Given that transcriptional control cannot be studied without considering chromatin as the real template for RNA polymerase, expecially because in many cases key promoter elements are occupied by nucleosomes (17Piña B. Brüggemeier U. Beato M. Cell. 1990; 60: 719-731Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 18Venter U. Svaren J. Schmitz J. Schmid A. Hözz W. EMBO J. 1994; 13: 4848-4855Crossref PubMed Scopus (118) Google Scholar, 19Steger D.J. Workman J.L. Bioessays. 1996; 18: 875-884Crossref PubMed Scopus (100) Google Scholar), many recent studies have concentrated on identifying the protein complexes capable of unfolding or modifying chromatin structure (20Kingston R.E. Bunker C.A. Imbalzano A. Genes Dev. 1996; 10: 905-920Crossref PubMed Scopus (404) Google Scholar, 21Griffin Burns L. Peterson C.L. Biochim. Biophys. Acta. 1997; 1350: 159-168Crossref PubMed Scopus (32) Google Scholar). These protein complexes have been found to act on specific genes and appear to be conserved among eukaryotic organisms (22Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 23Cairns B.R. Lorch Y. Li Y. Zhang M. Lacomis L. Erdjument-Bromage H. Tempst P. Du J. Laurent B. Kornberg R.D. Cell. 1996; 87: 1249-1260Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), suggesting that overcoming the nucleosomal barrier is indeed an important step in transcriptional activation. Nucleosomes have also been shown to possess special features that facilitate their recognition and aid in allowing access of the transcription initiation complex. Acetylation of specific histone residues have been implicated in targeted nucleosome disruption (24van Holde K.E. Lohr D.E. Robert C. J. Biol. Chem. 1992; 267: 2837-2840Abstract Full Text PDF PubMed Google Scholar, 25Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (477) Google Scholar, 26Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar) during activation processes, whereas the reverse reaction, deacetylation, appears to be required to ensure repression (27Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (718) Google Scholar,28Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). We have chosen as a model system the promoter of theSaccharomyces cerevisiae ADH2 gene coding for the enzyme alcohol dehydrogenase II (29Russel D.W. Smith M. Williamson V.M. Young E.T. J. Biol. Chem. 1983; 258: 2674-2682Abstract Full Text PDF PubMed Google Scholar) to answer two questions concerning the role of chromatin in gene activation. Is chromatin simply an obstacle for promoter recognition that once removed automatically leads to transcription? Given this context, what is the function of transcriptional activators? Previously, we have shown that a defined nucleosomal organization characterizes the ADH2 promoter in repressing (high glucose) conditions. Upon changing the medium to derepressing (low glucose) conditions, a modification occurs in the protection pattern of two nucleosomes that cover the basal transcription elements (TATA box and RNA initiation sites) (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar). This chromatin remodeling requires the product of the ADR1 gene (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar), coding for a zinc finger protein known to activateADH2 and genes involved in peroxisomal function (31Denis C.L. Young E.T. Mol. Cell. Biol. 1983; 3: 360-370Crossref PubMed Scopus (83) Google Scholar,32Simon M. Adam G. Rapatz W. Spevak W. Ruis H. Mol. Cell. Biol. 1991; 11: 699-704Crossref PubMed Scopus (132) Google Scholar). Genetic evidence indicates that at least two regulatory pathways are required for full ADH2 expression. Besides the transcriptional activator ADR1, which acts through the upstream activation sequence 1 region (33Shuster J. Yu J. Cox D. Chan R.V.L. Smith M. Young E. Mol. Cell. Biol. 1986; 6: 1894-1902Crossref PubMed Scopus (62) Google Scholar), a second pathway consisting of the activator CCR4 and the negative effectors SPT10 and SPT6 do not require specific upstream activation sequences (34Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (74) Google Scholar). Both activators are likely to interact with additional cofactors, ADR1 contacting ADA2 and GCN5, components of a histone acetylase complex (35Chiang Y.-C. Komarnitsky F. Chase D. Denis C.L. J. Biol. Chem. 1996; 271: 32359-32365Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), whereas CCR4 is part of a multisubunit transcriptional regulatory complex (34Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (74) Google Scholar). Here we investigate the role of different elements of theADH2 system (the positive factors CCR4 and ADR1, RNA polymerase II) by analysis of specific mutants to determine whether nucleosomal reconfiguration represents a necessary and sufficient event for promoter activation. We find that CCR4 is not required for altering chromatin structure but rather appears to control the transcription rate. ADR1, in contrast, is found to play a role in nucleosomal disruption even when this is induced by a signal (temperature increase) that does not lead to ADH2 mRNA accumulation. We also report that the catalytic activity of RNA polymerase II is not relevant for the maintenance of the chromatin alteration. As a whole, these results indicate that the modification of nucleosome structure at the ADH2 promoter is an important step in the activation process, although not sufficient for transcription. Yeast strains are listed in Table I. Strain RY260 is described in Ref. 36Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (287) Google Scholar. Strains HD93-15D and HD93-15DdB, kindly provided by G. Pereira and C. P. Hollenberg, are described in Ref. 37Pereira G.G. Hollenberg C.P. Eur. J. Biochem. 1996; 238: 181-191Crossref PubMed Scopus (20) Google Scholar.Table IYeast strainsStrainGenotypeEGY188MATa his3 leu2 trp1 ura3 lexAop-LEU2EGY188–1Same as EGY188 exceptccr4::URA3RY260MATa ura3–52 rpb1–1HD93–15DMATa his3 leu2 trp1 ura3HD93–15DbBSame as HD93–15D exceptadr1::HIS36–60MATa his4 trp1 ura1 adh1–11 adr1::ADR1-5c-TRP1R22.2–8BMATa ade2 trp2 ADH2–4 c Open table in a new tab Yeast cells were grown in 1% yeast extract, 2% peptone, 3% glucose as rich medium. ADH2 derepression was obtained as follows. Cells grown overnight in 200 ml of medium containing 3% glucose (to 0.5 optical density unit/ml) were washed with water and resuspended in the same volume of fresh medium containing 0.05% glucose. At different times, samples were collected, and both chromatin and RNA were analyzed. Temperature shifts were performed as follows. Cells grown overnight at 24 or 30 °C were pelleted and resuspended in the same volume of fresh medium equilibrated at 37 °C. The 2,250-base pair BamHI fragment shown in Fig. 1 and described in Ref. 30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar was inserted in theHindIII site of PUC18M (38Venditti S. Caserta M. Di Mauro E. Camilloni G. Biochim. Biophys. Acta. 1988; 951: 139-148Crossref PubMed Scopus (8) Google Scholar) to obtain plasmid pFA. This plasmid DNA was used to prepare probes for the indirect end-labeling analysis and as deproteinized material for control reactions with micrococcal nuclease (MN). 1The abbreviations used are: MN, micrococcal nuclease; MOPS, 4-morpholinepropanesulfonic acid. For the ADH2 sequence, see Ref. 29Russel D.W. Smith M. Williamson V.M. Young E.T. J. Biol. Chem. 1983; 258: 2674-2682Abstract Full Text PDF PubMed Google Scholar. All nucleases were purchased from Boehringer Mannheim, and Zymolyase 100T was purchased from Seikagaku Corp. The method is based on MN treatment of nystatin-permeabilized spheroplasts (39Venditti S. Camilloni G. Mol. Gen. Genet. 1994; 242: 100-104Crossref PubMed Scopus (30) Google Scholar). Cells (25–40 optical density units/sample) were pelleted and resuspended in 5–8 ml of Zymolyase buffer (1 m sorbitol, 50 mm Tris-HCl, pH 7.5, 10 mm 2-mercaptoethanol) in the presence of 0.05 mg of Zymolyase 100T/optical density unit. Incubation was carried out for 30 min at 30 °C. For the cells grown at 37 °C, incubation was at 37 °C. The pelleted spheroplasts were resuspended in nystatin buffer (50 mm NaCl, 1.5 mm CaCl2, 20 mm Tris-HCl, pH 8.0, 1 m sorbitol, 100 μg of nystatin/ml) and divided into 0.25 ml aliquots. MN (0.5–10 units) was added to each aliquot of permeabilized spheroplasts, and incubation was performed at 37 °C for 15 min. The reaction was stopped with 1% SDS and 5 mm EDTA (final concentrations). Proteinase K (40 μg/sample) was added, and the samples were kept at 56 °C for 2 h. The DNA was then purified by phenol-chloroform extractions (three times) and ethanol precipitation. In vitro treatment of samples with MN was as follows. Chromosomal or plasmid DNA was reacted in 0.1 ml of MN digestion buffer (50 mm NaCl, 1.5 mm CaCl2, 20 mm Tris-HCl, pH 8.0) with 0.25–2 units of MN for 3 min at 37 °C. The reaction was stopped with 1% SDS and 5 mm EDTA (final concentrations) and purified by phenol-chloroform extraction and ethanol precipitation. Indirect end-labeling analysis (40Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (775) Google Scholar) was performed as follows. After secondary digestion with BamHI and/or HindIII, the samples were electrophoresed in 1.5% agarose-Tris borate-EDTA (TBE) gels (1.75 V/cm), transferred to nitrocellulose paper (Schleicher & Schuell), and hybridized with probes 5′ or 3′, respectively, by standard procedures. For each experiment, aliquots containing an equal number of cells (0.5–1 × 108) were pelleted, and total RNA was prepared as described in Ref. 41Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1167) Google Scholar. After spectrophotometric determination of the amount of RNA present in each aliquot, 10–20 μg of RNA were loaded onto 1.2% agarose-MOPS gels containing formaldehyde as a denaturing agent and ethidium bromide as an intercalating dye. Northern blot analysis was performed by standard procedures with Hybond N+ nylon paper (Amersham). For hybridization, a 5′-end-labeled oligonucleotide specific for the ADH2 gene was used. Map positions and sequence are as follows. From +710 to +684, 5′-GTTGGTAGCCTTAACGACTGCGCTAAC-3′. The structure of the ADH2 promoter and the positioning of the three relevant nucleosomes (−2, −1, and +1, numbered relative to the RNA initiation sites) are shown in Fig.1. The borders of the multiple alternative particles have been determined by in vivo high resolution mapping on isolated monomeric DNA (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar). The chromatin organization presented in Fig. 1 characterizes the ADH2promoter in repressing (high glucose, 3%) conditions. Upon changing the medium to derepressing (low glucose, 0.05%) conditions, the two nucleosomes covering the TATA box and the RNA initiation sites (−1 and +1, respectively) underwent a rearrangement, causing loss of protection of the underlying sequences (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar). To determine which proteins (e.g. general or specific transcription factors, RNA polymerase II) were involved in this remodeling event, we have analyzed the ADH2 promoter chromatin structure in various genetic backgrounds. The assay used for all the experiments consists of MN digestion of nystatin-permeabilized spheroplasts from repressed or activated cells. After a secondary digestion with the appropriate restriction enzyme, the samples were analyzed by the indirect end-labeling technique (40Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (775) Google Scholar). Northern analysis was used to test the level of ADH2 mRNA accumulation in every mutant. One of the factors known to be involved inADH2 transcription is the product of the CCR4gene coding for a glucose-regulated protein, originally identified as a suppressor of mutations in the SPT6 and SPT10genes that allow increased transcription at ADH2 (42Denis C.L. Genetics. 1984; 108: 833-843Crossref PubMed Google Scholar). Given that SPT6 has been implicated in nucleosome assembly (43Bortvin A. Winston F. Science. 1996; 272: 1473-1476Crossref PubMed Scopus (297) Google Scholar), we wanted to analyze the role of CCR4 in chromatin organization. We analyzed ADH2 chromatin structure in two isogenic strains, one of which carried a CCR4 gene disruption. The results are shown in Fig. 2. In repressing conditions (Fig. 2 A, G = 3% glucose) in both the wild-type and the ccr4 mutant strains, the area occupied by the nucleosomes −1 and +1 was protected (small filled circles point out the strong MN cleavage sites found on naked DNA approximately at the level of the TATA box and the ATG consensus sequences; they are covered in the samples from repressed cells). When the cells were grown in derepressing conditions (Fig. 2 A,E = 3% ethanol) or shifted to low glucose (Fig.2 B, 0.05%G), loss of protection was observed for both strains, indicating that the two nucleosomes became modified. Two additional nucleosomes, −2 and +2, as previously reported (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar), became modified (Fig. 2 A, lanes E). The extent and the kinetics of this promoter modification were found to be very similar in both strains (compare, for example, Fig. 2 B, lanes 1 h at 0.05%G ). By contrast, the amount of steady state mRNA was substantially lower in the ccr4 mutant as compared with the wild-type strain, both in ethanol (Fig.2 C) and in low glucose (0.05%G, Fig.2 D) conditions. Interestingly, the kinetics ofADH2 mRNA accumulation shown in Fig. 2 Dindicated that ADH2 mRNA appears earlier in the absence of CCR4 (30 min time point), after which it slightly increases, never reaching the abundance observed in the wild-type strain. We conclude from these experiments that CCR4 is required downstream of the chromatin remodeling event to allow efficient transcription. The experiments reported in Fig. 2 show that the transcription rate is not correlated with the extent of nucleosome reconfiguration. In fact, the structural chromatin change occurs with the same efficiency irrespective of the mRNA amount produced. To further test the role of transcription in the remodeling process, we analyzed the chromatin structure at theADH2 promoter in an rpb1–1 strain containing an RNA polymerase II temperature-sensitive mutation (36Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (287) Google Scholar). When the temperature is increased to 37 °C in strains carrying this mutation, a block in the elongation ability of RNA polymerase II immediately occurs (36Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (287) Google Scholar). This mutation impairs the catalytic activity of the enzyme but does not influence the assembly of the subunits. Fig.3 A shows the results of this analysis. At the permissive temperature (24 °C) and in repressing conditions (3% glucose), the nucleosomes −1 and +1 were found to protect the ADH2 promoter. Surprisingly, the chromatin structure was found to be modified after 1 h of incubation at the nonpermissive temperature (37 °C), even under glucose-repressing conditions (lanes 3% G, 37 °C). If the cells were then washed and incubated in 0.05% glucose-containing medium at the same restrictive temperature, the chromatin remained open, whereas no trace of ADH2mRNA was observed (see Fig. 3 B). Two main facts emerge from this experiment. (i) Transcriptional elongation is not required to actively maintain the nucleosomes in the modified state; on the other hand, assembly of the pre-initiation complex, which is not impaired in the rpb1–1 strain at 37 °C, may still be responsible for the nucleosomal modification, and (ii) an environmental stimulus (heat), not apparently related to glucose metabolism, is able to induce the chromatin change at theADH2 promoter. To exclude the possibility that the nucleosome modification shown in Fig. 3 A(37 °C, 3% G) occurred fortuitously during the inactivation of the RNA polymerase II catalytic subunit, we analyzed the effect of temperature on chromatin remodeling in a wild-type strain containing a functional enzyme and, at the same time, we asked whether this effect requires the major ADH2 activator protein, ADR1. The results are shown in Fig. 4. Cells grown overnight at 30 °C in repressing conditions (3% glucose) were pelleted and resuspended in fresh medium equilibrated at 30 °C or 37 °C. After incubation at these two temperatures for 1 h without changing the glucose content of the medium, the chromatin was analyzed using the 3′ probe. The two bands indicating loss of nucleosome protection became visible only in the case of the wild-type strain, suggesting that the temperature-induced chromatin remodeling occurs also in a normal strain and requires ADR1. The result was confirmed using the 5′ probe (data not shown). We then asked whether this temperature-induced effect represents an alternative way to induce ADH2 expression in the wild-type strain, and we found that this is not the case. Fig.5 shows that no ADH2 mRNA accumulation was observed even after 3 h of incubation at 37 °C, if the cells were kept in 3% glucose. By contrast, when the cells incubated at 37 °C for 1 h in 3% glucose were shifted to 0.05% glucose-containing medium at the same temperature and tested at 1 and 3 h, ADH2 mRNA was detected. The kinetics of mRNA accumulation at this temperature were different from what is normally observed when cells are shifted to 0.05% glucose at the temperature of 30 °C (30Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell. Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (87) Google Scholar); here, the steady state mRNA level peaked at 1 h and thereafter decreased. These results indicate that the chromatin structure of theADH2 promoter is affected by at least two different signals: glucose reduction, which leads to transcriptional activation, and temperature increase, which does not. In addition, we observe that transcription is not required for chromatin remodeling at this promoter. Under two circumstances, when there is absolutely no ADH2 transcription (shift to 37 °C under glucose growth conditions and derepression in the presence of the defective rpb1–1 allele; see above), nucleosomal remodeling occurs and is maintained. The results shown in Figs. 4 and 5 imply that, in the presence of high glucose, ADR1 induces the nucleosome modification if the temperature is raised, but its function as a transcriptional activator is still repressed. We analyzed a mutant strain carrying the ADR1–5 c allele, which allows partially constitutive expression of the ADH2gene because of an Arg to Lys substitution in position 228 in ADR1 (44Denis C.L. Gallo C. Mol. Cell. Biol. 1986; 6: 4026-4030Crossref PubMed Scopus (33) Google Scholar). Fig. 6 shows the ADH2chromatin structure in the ADR1–5 c strain grown in 3% glucose. The ADH II activity in this strain is already high in 3% glucose and becomes higher than a corresponding wild type when the glucose is lowered (45Ciriacy M. Mol. Gen. Genet. 1979; 176: 427-431Crossref PubMed Scopus (91) Google Scholar). It can be seen from Fig. 6 that the chromatin structure of the promoter was disrupted under glucose growth conditions, suggesting that remodeling and transcription can be achieved in high glucose and at the normal temperature as long as the repression exerted on a specific domain of ADR1 is abolished. The first event in the transcriptional regulation of any gene is the processing of extracellular and/or intracellular signals, whereas the last phase consists of changes in the rate of transcription. To understand all the processes that happen in between, both in vitro and in vivo approaches need to be utilized. We have used a strategy of genetic dissection to analyze one particular moment in the process of transcriptional activation: the chromatin modification occurring at the S. cerevisiae ADH2 promoter when the cells are shifted from repressing to derepressing conditions of growth. This study has been carried out in different genetic backgrounds with two types of assays: indirect end-labeling analysis of MN-treated spheroplasts to look at specific nucleosomes and Northern analysis to check the extent of mRNA accumulation in the same cells from which spheroplasts are prepared. The rationale of this work was the examination of mutants belonging to different groups of proteins to identify those involved in the chromatin remodeling event. The results presented in this work outline a model for ADH2promoter activation in which at least two separate steps can be identified, as shown in Fig. 7. In the first step (repression → derepression), the structure of the nucleosomes −2, −1, and +1 located on the relevant promoter elements undergoes a remodeling event characterized by loss of protection of the underlying sequences. For this event to occur, the transcriptional activator ADR1 is required, and the repression exerted on the protein must be relieved. This can be achieved by several means: (i) by lowering the amount of glucose in the medium, (ii) by increasing the temperature from 30 to 37 °C, or (iii) by mutating ADR1 in the 227–239 region (46Denis C.L. Fontaine S.C. Chase D. Kemp B.E. Bemis L.T. Mol. Cell. Biol. 1992; 12: 1507-1514Crossref PubMed Scopus (39) Google Scholar). The catalytic activity of RNA polymerase II is not required for ADR1-mediated nucleosomal rearrangement (Fig. 3), whereas it is likely that the binding of the basal transcription factors and/or the assembly of the holoenzyme are involved because of the dependence on ADR1. Recent evidence suggesting an interaction between ADR1 and TFIIB supports a role for ADR1 in recruiting the transcriptional machinery (35Chiang Y.-C. Komarnitsky F. Chase D. Denis C.L. J. Biol. Chem. 1996; 271: 32359-32365Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The requirement of the transactivation domain of the activator protein Pho4 in the remodeling process occurring at the PHO5 promoter (47Svaren J. Schmitz J. Hözz W. EMBO J. 1994; 13: 4856-4862Crossref PubMed Scopus (110) Google Scholar) and the fact that a single contact with the RNA polymerase II holoenzyme is sufficient for the remodeling at that promoter (48Gaudreau L. Schmid A. Blaschke D. Ptashne M. Hözz W. Cell. 1997; 89: 55-62Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) is in line with this evidence. In the second step (derepression → transcription), transcription commences and, in the presence of the positive factors CCR4 and presumably ADR1, leads to full-level mRNA accumulation, whereas the chromatin remains in the open configuration. The two-step model outlined for the ADH2 promoter has been already established in vivo for other yeast genes likeSUC2 (49Hirshhorn J.N. Brown S.A. Clark C.D. Winston F. Genes Dev. 1992; 6: 2288-2298Crossref PubMed Scopus (442) Google Scholar), PHO5 (50Fasher K.-D. Schmitz J. Hözz W. J. Mol. Biol. 1993; 231: 658-667Crossref PubMed Scopus (99) Google Scholar), and GAL1 (51Selleck S.B. Majors J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5399-5403Crossref PubMed Scopus (21) Google Scholar) by using TATA box mutants to affect the transcription level. However, in the case of the ADH2 promoter (this work), the TATA box is integral, and all the other promoter elements have been retained in their normal chromosomal location. In no case do we observe transcription without nucleosome disruption. On the other hand, the chromatin remodeling by itself is not sufficient to allow maximal level of ADH2 mRNA accumulation. In fact, we find that (i) the heat-induced chromatin remodeling is not followed by ADH2 mRNA synthesis as long as the glucose content of the medium is kept high (Figs. 4 and 5), and (ii) in the absence of CCR4, the ADH2 mRNA level is low even though the extent and the kinetics of nucleosome disruption do not differ from a wild-type isogenic strain (Fig. 2). According to the working model shown in Fig. 7, we predict that when the glucose level becomes sufficiently low ADR1 primes the assembly of the pre-initiation complex, and at the same time, the disruption of the two nucleosomes (−1 and +1) protecting the TATA box and the RNA initiation sites (Fig. 7, first step). The assembly of the pre-initiation complex proceeds via initial recruitment of TFIID and subsequent recruitment of the holoenzyme, as recently proposed (52Stargell L.A. Struhl K. Trends Genet. 1996; 12: 311-315Abstract Full Text PDF PubMed Scopus (103) Google Scholar). The fact that part of the TFIID structure closely resembles the histone octamer structure (53Surridge C. Nature. 1996; 380: 287-288Crossref PubMed Scopus (1) Google Scholar, 54van Holde K. Zlatanova J. Bioessays. 1996; 18: 697-700Crossref PubMed Scopus (16) Google Scholar) suggests a speculative model of molecular substitution. In the second step (Fig. 7), transcriptional elongation takes place only if additional negative effectors are removed; part of this repression is exerted again on ADR1 and part on CCR4. The transcriptional activator CCR4 is, in fact, glucose-regulated and has been found to be part of a multisubunit transcriptional regulatory complex (34Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (74) Google Scholar, 55Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Crossref PubMed Scopus (96) Google Scholar). More recently, a functional interaction between the CCR4 complex and a cell cycle-regulated protein kinase has been reported (56Liu H.-Y. Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (89) Google Scholar). Perhaps CCR4 contacts some component of the holoenzyme and introduces a stabilizing effect. In fact, in its absence, the sudden increase in ADH2mRNA accumulation that occurs in a wild-type strain after the start of transcription is not observed (Fig. 2, panel D). The known genetic interaction of SPT6 and CCR4 is also clarified by the data presented herein. Mutations in CCR4 and CAF1(a component of the CCR4 complex) (55Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Crossref PubMed Scopus (96) Google Scholar) are the only known mutations that reduce the ability of spt6 defects to allow increased transcription (42Denis C.L. Genetics. 1984; 108: 833-843Crossref PubMed Google Scholar, 55Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Crossref PubMed Scopus (96) Google Scholar, 57Denis C.L. Malvar T. Genetics. 1990; 124: 283-291Crossref PubMed Google Scholar). SPT6 has been shown to bind histones and, when mutated, to result in nucleosomal rearrangement (43Bortvin A. Winston F. Science. 1996; 272: 1473-1476Crossref PubMed Scopus (297) Google Scholar). The increased transcription resulting from spt6 mutations is therefore consonant with its effects on nucleosomal rearrangement. Once this rearrangement occurs there may be several different pathways for activation, perhaps depending on how the transcriptional initiation complex is stabilized. Alternatively, the chromatin conformation at theADH2 promoter caused by mutant alleles such asspt6 and ADR1–5 c or by other factors like glucose or high temperature may all not be physically identical. We have also found that the ADR1 protein is required for the heat-induced chromatin disruption (Fig. 4). This result is intriguing; the same protein appears to be the target of two different extracellular signals. In looking for a common feature to these signals, it may be possible that glucose decrease in the medium is a stress just as temperature increase. Different stress conditions might trigger a general response by creating the same intracellular signal (for a discussion see Ref. 58Ruis H. Schüller C. Bioessays. 1995; 17: 959-965Crossref PubMed Scopus (364) Google Scholar). We are currently trying to understand whether ADR1 can work as a mediator of stress effects (to be detailed elsewhere). The two events leading to chromatin remodeling (low glucose and temperature increase) can follow two different pathways, converging on the same molecule (ADR1) but involving different domains of the protein. Alternatively, the pathway is the same; the two extracellular signals are transduced very soon to the same molecule, which leads to the ADR1-dependent nucleosome change, but glucose still controls the transcriptional ability of the activator. Evidence for multiple levels of control of ADR1 have indeed been presented (59Vallari R.C. Cook W.J. Audino D.C. Morgan M.J. Jensen D.E. Laudano A.P. Denis C.L. Mol. Cell. Biol. 1992; 12: 1663-1673Crossref PubMed Scopus (45) Google Scholar). It cannot be excluded, however, that the two chromatin modifications are structurally different. High resolution analysis will help to clarify this point. We thank M. Marchioni for helping to perform some of the experiments and A. Di Francesco, R. Gargamelli, and G. Ricci for technical help.