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• W1987928196 abstract "Analysis of the chromatin structure at the yeastILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region. All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA·dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site. The existence of a second A·T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA·dT) elements. Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription. Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter. The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence. Analysis of the chromatin structure at the yeastILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region. All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA·dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site. The existence of a second A·T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA·dT) elements. Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription. Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter. The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence. subtelomeric anti-silencing regions hypersensitive site 5-methyl-dl-tryptophan The Saccharomyces cerevisiae anabolic threonine deaminase, encoded by the ILV1 gene, catalyzes the first committed step in the isoleucine biosynthetic pathway. ILV1basal level expression (defined as the level of expression observed in a Δgcn4 strain grown in minimal medium) is controlled by two cis-acting elements: a binding site for the REB1 protein (Reb1p) and a poly(dA·dT) element (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar, 2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). Reb1p (Grf2, Qbp, factor Y or Q) is an essential, abundant DNA-binding protein with numerous binding sites present throughout the genome, many of which are located within regulatory regions for RNA polymerase II transcribed genes. Other Reb1p-binding sites are found in RNA polymerase I regulatory regions and in diverse genetic elements, such as centromeres and telomeres (3Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (163) Google Scholar, 4Ju Q. Morrow B.E. Warner J.R. Mol. Cell. Biol. 1990; 10: 5226-5234Crossref PubMed Google Scholar, 5Morrow B.E. Johnson S.P. Warner J.R. Mol. Cell. Biol. 1993; 13: 1283-1289Crossref PubMed Scopus (31) Google Scholar, 6Lang W.H. Reeder R.H. Mol. Cell. Biol. 1993; 13: 649-658Crossref PubMed Scopus (87) Google Scholar, 7Dammann R. Lucchini R. Koller T. Sogo J.M. Nucleic Acids Res. 1993; 21: 2331-2338Crossref PubMed Scopus (236) Google Scholar). Reb1p-binding sites are also found in the recently identified STAR1 (subtelomeric anti-silencing regions) elements (8Fourel G. Revardel E. Koering C.E. Gilson E. EMBO J. 1999; 18: 2522-2537Crossref PubMed Scopus (199) Google Scholar). These regions function as insulators and can protect neighboring genes from surrounding silencing elements. Interestingly, tandemly repeated binding sites for Reb1p can duplicate this anti-silencing effect and limit telomeric silenced chromatin. Furthermore, Reb1p-binding sites can stimulate or diminish transcription in a context-dependent manner (9Brandl C.J. Struhl K. Mol. Cell. Biol. 1990; 10: 4256-4265Crossref PubMed Scopus (30) Google Scholar, 10Wang H. Nicholson P.R. Stillman D.J. Mol. Cell. Biol. 1990; 10: 1743-1753Crossref PubMed Scopus (45) Google Scholar, 11Brandl C.J. Martens J.A. Liaw P.C-Y. Furlanetto A.M. Wobbe C.R. J. Biol. Chem. 1992; 267: 20943-20952Abstract Full Text PDF PubMed Google Scholar). Although Reb1p affects transcription of RNA polymerase II transcribed genes, the mechanism by which it does so is not clear. The presence of a Reb1p-binding site in the GAL1-GAL10 promoter correlated with a nucleosome-free region of about 230 bp in vivo (12Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Crossref PubMed Scopus (184) Google Scholar) suggesting that Reb1p functions to generate a nucleosome-free region allowing auxiliary factors access to adjacentcis-acting elements. Other reports, however, contest this result (13Axelrod J.D. Reagan M.S. Majors J. Genes Dev. 1993; 7: 857-869Crossref PubMed Scopus (94) Google Scholar, 14Reagan M.S. Majors J.E. Mol. Gen. Genet. 1998; 259: 142-149Crossref PubMed Scopus (17) Google Scholar) casting some doubt as to the mechanism of action of Reb1p. Homopolymeric poly(dA·dT) sequences are present in the promoter region of many yeast genes and have been shown to influence transcription of several genes (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar, 15Chen W. Tabor S. Struhl K. Cell. 1987; 50: 1047-1055Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 16Lue N.F. Buchman A.R. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 486-490Crossref PubMed Scopus (75) Google Scholar, 17Thiry-Blaise L.M. Loppes R. Mol. Gen. Genet. 1990; 223: 474-480Crossref PubMed Scopus (10) Google Scholar, 18Iyer V. Struhl K. EMBO J. 1995; 14: 2570-2579Crossref PubMed Scopus (348) Google Scholar). Due to their nucleosome destabilizing properties poly(dA·dT) elements have been proposed to function by virtue of their intrinsic effect on chromatin (15Chen W. Tabor S. Struhl K. Cell. 1987; 50: 1047-1055Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 18Iyer V. Struhl K. EMBO J. 1995; 14: 2570-2579Crossref PubMed Scopus (348) Google Scholar). Interestingly, the yeast poly(dA·dT)-binding protein Dat1p (19Winter E. Varshavsky A. EMBO J. 1989; 8: 1867-1877Crossref PubMed Scopus (91) Google Scholar) functions as a transcriptional activator of ILV1 expression and this action depends on the presence of the poly(dA·dT) element (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). In an attempt to elucidate the mechanism by which the Reb1p-binding site and the downstream poly(dA·dT) element control ILV1basal expression, we investigated the chromatin structure of theILV1 locus. We show that the ILV1 promoter and coding regions are assembled into a highly ordered nucleosome array, with a single hypersensitive region encompassing all regulatorycis-acting elements. We also show that deletion of the Reb1p site and/or the adjacent downstream poly(dA·dT) element greatly diminishes ILV1 expression, yet does not cause reconfiguration of the ILV1 chromatin structure. Furthermore, a second A·T-rich element present upstream of the Reb1p-binding site can be deleted, again with no effect onILV1 chromatin structure. This suggests that neither Reb1p nor the adjacent poly(dA·dT) elements stimulate ILV1transcription by increasing the accessibility of DNA in chromatin at the promoter, but by another yet unknown mechanism. Sequence insertions and deletions at the hypersensitive region of the ILV1promoter, albeit affecting expression of the ILV1 gene, do not affect the positioning of the adjacent nucleosomes pointing to a dominant effect of the DNA sequence in organizing the nucleosomal array present at the ILV1 promoter. Escherichia colistrain DH5α was used for plasmid propagation and manipulation. The following S. cerevisiae strains have been used in this study: TD28 (MATaura3–52 ino1 can1), 9994–6C (MATα Δgcn4 ura3–52), EWY1002c-1 (MATalys2–801 leu2–3,112 ura3–52 his3-Δ200 trp1–1(am)), BRY1004–3 (MATa lys2–801 leu2–3,112 ura3–52 his3-Δ200 trp1–1(am) dat1-Δ2), TG561 (MATailv1::lacZ ino1 can1), TG570 (EWY1002c-1 ilv1::lacZ). Strains were grown in minimal medium (0.67% Bacto Yeast Nitrogen Base without Amino Acids, 2% glucose, buffered with 10 g of succinic acid and 6 g of NaOH per liter) supplemented with the required amino acids at appropriate concentrations. Restriction endonucleases and DNA-modifying enzymes were purchased fromRoche Molecular Biochemicals (Mannheim, Germany). Zymolyase 100T was from Seikagaku America, Inc. Taq polymerase was from Amersham Biosciences. Radiolabeled nucleotides were from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Agarose was from FMC Bioproducts (Rockland, ME). All nucleic acid manipulation was performed according to established protocols (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Polymerase chain reaction (PCR) was used under standard conditions (0.2 mm of each of dATP, dCTP, dTTP, dGTP; 20 mmTris-HCl (pH 8.4); 50 mm KCl; 5 mmMgCl2; 0.5 μm of each primer; 2.5 units ofTaq DNA polymerase per reaction). DNA was sequenced using the dideoxy primer extension method (Sequenase Sequencing Kit v2.0, United States Biochemical Corporation, Cleveland, Ohio). Yeast cells were transformed following the method of Ito et al. (21Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Northern analysis was performed as described (22Moreira J.M.A. Holmberg S. EMBO J. 1999; 18: 2836-2844Crossref PubMed Scopus (78) Google Scholar). Nuclei isolation and nuclease digestions were performed as previously described (23Svaren J. Venter U. Hörz W. Microbial Gene Techniques. Academic Press, New York1995Google Scholar). After secondary digestion with the appropriate restriction enzyme, DNA samples were electrophoresed in 1.5% agarose gels, transferred onto PositiveTM nylon membranes (Oncor, Gaithersburg, MD) and hybridized following standard protocols. A plasmid containing a 6.1-kbHindIII-SalI ILV1 fragment (pC519) was digested with the appropriate restriction enzymes and electrophoretically purified DNA fragments labeled with the Prime-It® II random primer labeling kit (Stratagene, La Jolla, CA) and used as probes in all experiments. The specific fragments used as probes are described in further detail in figure legends. For the spacing experiments, we used plasmid constructs containing insertions of E. coliplasmid DNA into the XhoI site of the 15X ILV1internal deletion fused to the lacZ gene (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). The constructs were integrated at the ILV1 locus in strain 9994–6C (Δgcn4). All integration events were confirmed by Southern analysis and PCR. Deletion of the ILV1 A·T-rich tracts was made by PCR using a previously described procedure (24Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). Oligonucleotides covering the sequences we wished to modify were synthesized (T·A·G Copenhagen ApS, Denmark) allowing the target sequences to be substituted by random sequences with no known cis-acting elements. Oligonucleotide SUBUP (5′-AATTGACGCGAACTAGATCGCTTAAGCTCGGCATCAAGCTCGAGCGCAGCGGGTAGCAAATTTGGAATCG-3′) was used to replace the 5′-most ILV1 A·T-rich tract (positions −235 to −166) while simultaneously creating a uniqueXhoI site. Similarly, oligonucleotide SUBDOWN (5′-ATCTGCAGACATATGTTTGAGATGACTCTAGATCTCCGATGTTCAAGCTTCATGATTATGCGATTCCAAATTTGC-3′) was used to replace the 3′-most ILV1 A·T-rich tract (positions −106 to −180) and simultaneously introduce aHindIII site. PCR fragments were obtained covering theILV1 promoter and entire coding region (from position −840 to position +2530) that contained a substitution of the 5′-most, the 3′ most, or both A·T-rich tracts in the promoter region. The amplificates were cloned into plasmid pGEM-TTM (Promega) and the substitutions confirmed by sequencing and subsequently integrated at the ILV1 locus in strain TG561 generatingILV1(ΔA1), ILV1(ΔA2), andILV1(ΔA1ΔA2), respectively. We have shown that two cis-acting sequences, a Reb1p-binding site and a downstream poly(dA·dT) element control ILV1basal-level (GCN4-independent) expression (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar, 2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). The two elements act synergistically, an indication that they might exert their effect trough a common activation pathway (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). Sequence inspection revealed an additional A·T-rich tract (25 As out of 33 nucleotides) located six bp upstream of the Reb1p-binding site. To determine whether the upstream A·T-rich tract also plays a role in ILV1basal expression we replaced either one or bothILV1 A·T-rich tracts by 40% GC content random sequences resulting in strains ILV1(ΔA1),ILV1(ΔA2), and ILV1(ΔA1ΔA2), respectively. Northern analysis showed that both A·T-rich tracts potentiateILV1 basal expression (Fig.1). We analyzed the nucleosomal organization of theILV1 promoter using digestion of yeast nuclei with DNase I, micrococcal nuclease (MNase), or restriction enzymes to map nuclease cuts by indirect end-labeling (25Wu C. Wong Y.C. Elgin S.C. Cell. 1979; 16: 807-814Abstract Full Text PDF PubMed Scopus (268) Google Scholar). Thus, nuclei from strain TD28 (GCN4) were treated with DNase I (see “Materials and Methods”), and the isolated DNA was resolved in an agarose gel after digestion with EcoRI, blotted, and hybridized with a radioactively labeled EcoRI-DraI probe. The obtained DNase I pattern (Fig.2) shows a characteristic ladder of bands typical of an ordered nucleosomal array, and a strong band corresponding to a hypersensitive site (HS). Mapping of this HS revealed that all previously identified cis-acting elements, namely a Reb1p-binding site, a Gcn4p-binding site, and the two poly(dA·dT) elements were located within the hypersensitive region. To complement the DNase I analysis and confirm our interpretation of the observed banding pattern, we digested nuclei from TD28 cells with various restriction enzymes (Fig. 3,A and B). Hypersensitive sites and linker regions are expected to be susceptible to digestion with endonucleolytic enzymes, whereas sequences assembled into nucleosomes should be protected. Very strong, nucleosomal sites are resistant to cleavage over a large range in enzyme concentration while non-nucleosomal sites are cut at much lower concentrations. Consequently accessibility reaches plateau levels both for nucleosomal and non-nucleosomal sites. To be certain in our experiments that we have truly reached these plateau values for a given site, we always verify that 3- to 4-fold higher nuclease concentrations still give the same accessibility thus ruling out that the enzyme activity had been limiting. Nuclei from yeast strain TD28 (GCN4) were digested withPvuII, BstUI, DraI, HinfI, or PstI at 500 and 1500 units/ml for 1 h. The results shown in Fig. 3, A and B confirm our conclusions derived from the DNase I assay. Bands were quantified using a phosphorimager equipped with ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA), and percent accessibility for the various enzymes was calculated from the ratio between the bands corresponding to digested sites and that of undigested DNA. Thus, the twoPstI sites present in the promoter region showed 50% (position −108) and <5% (position −546) accessibility, consistent with being at the border of the HS and within a nucleosome, respectively. BstUI (position −227) displayed 75% accessibility, correlating well with a position within the hypersensitive region. Conversely, the two DraI sites (positions −364 and −28) were less than 5% accessible, consistent with a location within the nucleosomes flanking the HS.PvuII (position −432) with 30% accessibility locates to an internucleosomal region in the deduced ILV1 chromatin structure. Five HinfI sites were examined (positions −595, −486, −296, −170, and −132). The sites within the hypersensitive region showed 80% accessibility (positions −170 and −132), whereas the remaining sites displayed less than 5% accessibility, consistent with nucleosomal locations. These results confirm the DNase I analysis and the mapping of nucleosomal positions we derived from that analysis. We conclude that the ILV1 promoter is organized in an ordered nucleosomal array with one strong hypersensitive site encompassing the region where the UAS elements are located. A schematic drawing of the ILV1 locus with restriction sites,cis-acting elements, and inferred chromatin structure is shown in Fig. 3C. The S. cerevisiae ILV1 gene is under regulation of the general control of amino acid biosynthesis. A Gcn4p-binding site present at position −127 was found to bind Gcn4p in vitro (26Arndt K.T. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8516-8520Crossref PubMed Scopus (190) Google Scholar). Indeed, upon amino acid starvation ILV1 expression is increased 2-fold by the Gcn4p activator protein (27Holmberg S. Petersen J.G.L. Curr. Genet. 1988; 13: 207-217Crossref PubMed Scopus (43) Google Scholar). We tried to determine whether derepression by Gcn4p had any effect on ILV1 chromatin structure. For derepression by the general control of amino acid biosynthesis, the tryptophan analog 5-methyl-dl-tryptophan (MeTrp) was added at a final concentration of 0.5 mm to the growth medium (27Holmberg S. Petersen J.G.L. Curr. Genet. 1988; 13: 207-217Crossref PubMed Scopus (43) Google Scholar, 28Miozzari G. Niederberger P. Hütter R. J. Bacteriol. 1978; 134: 48-59Crossref PubMed Google Scholar). Nuclei were prepared from TD28 (GCN4) cells grown to a density of 3 × 106 cells/ml and subjected to nuclease digestion with micrococcal nuclease (MNase) and DNase I, enabling us to complement our structural analysis. RNA was isolated from non-digested nuclei, and Northern analysis performed to confirm that ILV1 expression increased 2-fold upon derepression by the general control of amino acid biosynthesis (data not shown). The pattern obtained with either MNase (Fig.4) or DNase I (data not shown) was identical to the one previously observed for the basal transcriptional state of the ILV1 gene with a strong hypersensitive site comprising the UAS elements and an ordered nucleosomal array covering the promoter and coding region. Hence, derepression of theILV1 gene does not modify its chromatin structure in a way detectable in our analyses. We investigated the role of twotrans-acting factors, Dat1p and Gcn4p, both of which have been shown to bind their cognate sites at the ILV1 promoterin vitro (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar, 26Arndt K.T. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8516-8520Crossref PubMed Scopus (190) Google Scholar). Nuclei isolated from strains TD28 (GCN4), 9994–6C (Δgcn4), EWY1002c-1 (DAT1), and BRY1004–3 (Δdat1) grown in minimal medium were digested with DNase I, DNA was isolated, and end-labeling analysis performed as described above. The patterns obtained for all four strains were identical (data not shown), demonstrating that neither Gcn4p nor Dat1p are involved in generating the observedILV1 chromatin structure. A Reb1p-binding site present at position −180 is required forGCN4-independent ILV1 basal level transcription. Deletion of the Reb1p site reduces ILV1 basal level expression 10- to 15-fold (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar). Additionally, the poly(dA·dT) element located between positions −164 and −133, which is also necessary for wild-type ILV1 basal level expression (see above), is situated 15 bp downstream of the Reb1p site, and the two elements cooperatively stimulate ILV1 transcription (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). This synergistic activation could be the result of a common mechanism of action. A Reb1p-binding site in the GAL1-GAL10 promoter was correlated with the presence of a nucleosome-free region of about 230 bp (12Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Crossref PubMed Scopus (184) Google Scholar). Furthermore, poly(dA·dT) sequences stimulate Gcn4p-activated transcription and were suggested to function by changing chromatin structure and increasing accessibility of adjacent Gcn4p sites (18Iyer V. Struhl K. EMBO J. 1995; 14: 2570-2579Crossref PubMed Scopus (348) Google Scholar). Thus, the cooperative activation by the ILV1 Reb1p site and the downstream poly(dA·dT) element could be due to interactions between these two elements leading to increased accessibility of the adjacent Gcn4p-binding site. To test this possibility, we analyzed previously characterized promoter constructs showing decreasedILV1 basal expression (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). In these constructs theILV1 coding region has been replaced by the E. coli lacZ gene encoding β-galactosidase (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar). To determine whether the replacement of the ILV1 coding region with lacZ affected the structure of theILV1 promoter, we compared MNase digests of strain TD28 with those of strain TG561, an isogenic TD28 derivative containing aILV1 wild-type promoter, but with the ILV1 coding sequence replaced by lacZ. The digestion pattern for strain TD28 (Fig. 5, ILV1) is the expected one, a strong HS located in the promoter region, and an ordered nucleosomal array covers the coding region and promoter. Strain TG561 where the ILV1 coding sequence was substituted withlacZ shows a different pattern (Fig. 5, lacZ). The ILV1 promoter conserved its structure with one exception: the hypersensitive site was slightly decreased in size. This appears to be due to a shifted position of the nucleosome positioned at the downstream boundary of the hypersensitive region. Moreover, thelacZ sequence appears strongly nuclease-sensitive suggesting this gene to be less prone to undergo an ordered nucleosome organization. To confirm these conclusions and to determine that no accessibility changes had occurred in the hybrid promoter as compared with the wild-type promoter, we performed restriction enzyme analysis on the two strains. The results (Fig. 6) show that BstUI accessibility is almost identical (75% inILV1 and 70% in lacZ) but that thePstI site at position −108 changed from 50% accessibility in the wild-type situation (Fig. 6, ILV1) to <5% accessibility in the hybrid construct (Fig. 6, lacZ), consistent with going from a location at the border of the hypersensitive site to being covered by the adjacent nucleosome. The original position of this nucleosome had incorporated about 20 bp of coding sequence. Apparently, in the lacZ derivative, this positioned nucleosome is shifted upstream by about 20 bp thus excluding the new coding sequence altogether. At any rate, we conclude that the structure of the hybrid promoter construct reflects the wild-type situation in a sufficiently adequate manner for our study.Figure 6Accessibility analysis of the ILV1promoter in strains TD28 (wild-type) and TG561 (chimericILV1-lacZ fusion). Restriction enzyme analysis was performed as described (Fig. 3 legend). Position −108 corresponding to a PstI recognition site with altered accessibility is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We carried out a DNase I analysis on four constructs, which had previously been used to identify the Reb1p site and the downstream poly(dA·dT) element as important basal regulatory elements in theILV1 promoter (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar, 2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). The promoter derivatives are either deleted for the Reb1p-binding site and/or the poly(dA·dT) element. Again, the pattern observed is identical in all three derivatives and indistinguishable from the wild-type pattern (Fig.7, A and B and data not shown) suggesting that neither the Reb1p-binding site nor the poly(dA·dT) are responsible for generating of the HS present at theILV1 promoter. To rule out the possibility that deletion of the Reb1p-binding site or/and the neighboring poly(dA·dT) element has more subtle effects on chromatin not detected by DNase I digestion, we performed a restriction enzyme analysis of three of the constructs as well (Fig. 7C). BstUI (position −227) accessibility is similar in the tested constructs (75–70%), and thePstI site at position −108, as shown before, displayed <5% accessibility (Fig. 7C, ΔREB1Δ(dA·dT)) in the lacZ constructs. These results show that neither the ILV1 Reb1p-binding site nor the downstream poly(dA·dT) element are responsible for generating the hypersensitive region we observe in the ILV1 promoter. We have shown that ILV1 basal level expression depends on the distance separating the Reb1p-binding site and the downstream poly(dA·dT) element, since insertion of spacing DNA into aXhoI site created between the Reb1p-binding site and the poly(dA·dT) element in the 15X construct reduces promoter activity (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). We reasoned that if the hypersensitive site in the ILV1promoter were caused by any cis-acting element present within the nuclease accessible region, insertion of 41 bp or 74 bp of DNA corresponding to a size increase of 22% and 41%, respectively, might cause some rearrangement of the borders of the hypersensitive region thus affecting promoter activity. DNase I analysis of the constructs containing insertions of DNA between the Reb1p-binding site and the downstream poly(dA·dT) element is shown in Fig. 8. The basic structure and relative positioning of nucleosomes is preserved in the constructs containing insertions relative to the wild-type situation. The main difference is the progressive increase in the size of the nuclease-sensitive region. The resulting pattern clearly shows that the entire promoter region structure is similar between all the constructs and that insertion of increasingly larger spacing DNA is compensated for by correspondingly larger hypersensitive regions. We considered the possibility that the presence of two A·T-rich tracts placed symmetrically within the HS and located at roughly the same distance from the borders of the HS triggers nucleosome exclusion from this region. Such a mechanism would also explain the lengthening of the hypersensitive site observed as a consequence of introducing random sequences in between the two A-T rich tracts. We therefore replaced either one or both ILV1 A·T-rich tracts by 40% GC content random sequences resulting in strainsILV1(ΔA1), ILV1(ΔA2), andILV1(ΔA1ΔA2), respectively. MNase analysis of these strains is shown in Fig. 9. The obtained pattern is identical to the one previously detected in theILV1 promoter. Neither the typical nucleosomal ladder nor the hypersensitive region shows any significant difference to the wild-type situation. We conclude that the strong nucleosome positioning which we observe at the ILV1 locus is not dependent on the presence of the two A·T-rich tracts present in the promoter region. We have reported that ILV1 basal level expression is controlled in a synergistic manner by a Reb1p-binding site and a downstream poly(dA·dT) element (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). Reb1p has been proposed to antagonize nucleosomal repression by creating an accessible chromatin structure, thereby allowing other factors to gain access to their cognate sites (12Fedor M.J. Lue N.F. Kornberg R.D. J. Mol. Biol. 1988; 204: 109-127Crossref PubMed Scopus (184) Google Scholar). Other reports, however, dispute this conclusion and interpret the data differently (13Axelrod J.D. Reagan M.S. Majors J. Genes Dev. 1993; 7: 857-869Crossref PubMed Scopus (94) Google Scholar, 14Reagan M.S. Majors J.E. Mol. Gen. Genet. 1998; 259: 142-149Crossref PubMed Scopus (17) Google Scholar). Reb1p has also been implicated as a regulatory factor both in Pol II and Pol I transcribed genes (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar, 6Lang W.H. Reeder R.H. Mol. Cell. Biol. 1993; 13: 649-658Crossref PubMed Scopus (87) Google Scholar,9Brandl C.J. Struhl K. Mol. Cell. Biol. 1990; 10: 4256-4265Crossref PubMed Scopus (30) Google Scholar). Synergistic interactions between Reb1p-binding sites and poly(dA·dT) elements have been described by several authors (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar, 3Chasman D.I. Lue N.F. Buchman A.R. LaPointe J.W. Lorch Y. Kornberg R.D. Genes Dev. 1990; 4: 503-514Crossref PubMed Scopus (163) Google Scholar, 29Graham I.R. Chambers A. Mol. Microbiol. 1994; 12: 931-940Crossref PubMed Scopus (21) Google Scholar, 30Buchman A.R. Kornberg R.D. Mol. Cell. Biol. 1990; 10: 887-897Crossref PubMed Scopus (107) Google Scholar), raising the possibility that the two elements cooperate to increase accessibility of adjacent elements since also homopolymeric poly(dA·dT) elements have been proposed to stimulate transcription through an effect on chromatin structure (15Chen W. Tabor S. Struhl K. Cell. 1987; 50: 1047-1055Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 18Iyer V. Struhl K. EMBO J. 1995; 14: 2570-2579Crossref PubMed Scopus (348) Google Scholar). Given the presence of a Reb1p-binding site and two poly(dA·dT) elements in theILV1 promoter and the fact that at least one poly(dA·dT) element synergizes with Reb1p to sustain a relatively high basal level expression of the ILV1 gene, it is possible that the two elements act to increase chromatin accessibility, perhaps by keeping the promoter free from nucleosomes. To ascertain if Reb1p or the poly(dA·dT) elements have an effect on chromatin at ILV1, we examined the structure of theILV1 locus and mapped the nucleosomal of this gene (Fig.3C). The promoter and coding region are covered by an ordered nucleosomal array that is interrupted by a single strong nuclease-sensitive site comprising the region where thecis-acting elements required for normal expression of theILV1 gene are located. The structure of the locus is unaltered by Gcn4p-mediated derepression (Fig. 4). This is not an unexpected result since ILV1 basal level expression is relatively high and ILV1 is up-regulated only 2-fold by amino acid starvation (26Arndt K.T. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8516-8520Crossref PubMed Scopus (190) Google Scholar, 27Holmberg S. Petersen J.G.L. Curr. Genet. 1988; 13: 207-217Crossref PubMed Scopus (43) Google Scholar). Additionally, neither eliminations of Dat1p nor of Gcn4p, two trans-acting factors required for normal levels of expression, affected the chromatin structure of theILV1 locus (data not shown). It seemed reasonable to expect that Reb1p functions in theILV1 context through an effect on chromatin structure, establishing the nucleosome-free area of about 180 bp observed in the promoter region. However, constructs containing deletions in the Reb1p-binding site and/or the downstream poly(dA·dT) element, which decrease expression of the ILV1 gene or even abolish it (β-galactosidase measurements expressed in Miller Units; wild-type: 4.3 ± 0.8; ΔReb1: 0.51 ± 0.02; Δpoly(dA·dT); 0.36 ± 0.05; ΔReb1Δpoly(dA·dT): 0.09 ± 0.02) (1Remacle J.E. Holmberg S. Mol. Cell. Biol. 1992; 12: 5516-5526Crossref PubMed Scopus (37) Google Scholar, 2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar) showed the same chromatin structure as a wild-type promoter construct (Fig. 7, A–C), thus disproving the hypothesis that Reb1p is responsible for the hypersensitive site in the ILV1promoter. These data were obtained with ILV1-lacZ fusions replacing ILV1, and are therefore open to the criticism that they are not relevant for the native locus. In fact, the structure of the fusion constructs does not match perfectly the situation in the wild-type ILV1 locus, since the hypersensitive region is decreased by ∼20 bp (Fig. 5). Two lines of evidence argue against this criticism. First, quantitative analysis of accessibility in the region immediately upstream of the Reb1p-binding site did not show a significant difference between an ILV1 wild-type situation and the ILV1-lacZ fusion construct (Fig. 6). These data suggest that the observed decrease in the size of the hypersensitive site is a localized effect at the boundary to the lacZ coding region. Second, deletion of the Reb1p-binding site in theILV1-lacZ hybrid construct decreased transcription to about 10% of the wild-type levels as determined by β-galactosidase activity (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). A decrease in promoter accessibility having such a dramatic effect on gene expression would be expected to be easily revealed by the restriction enzyme analysis presented in Fig.7C, if it existed. However, no difference was detected. We conclude that Reb1p is not responsible for the formation of the hypersensitive site present in the ILV1 promoter region and that Reb1p potentiates expression in the ILV1 context by a mechanism other than causing a nucleosome-free region. Poly(dA·dT) elements stimulate transcription of several yeast genes (15Chen W. Tabor S. Struhl K. Cell. 1987; 50: 1047-1055Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 16Lue N.F. Buchman A.R. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 486-490Crossref PubMed Scopus (75) Google Scholar, 17Thiry-Blaise L.M. Loppes R. Mol. Gen. Genet. 1990; 223: 474-480Crossref PubMed Scopus (10) Google Scholar, 18Iyer V. Struhl K. EMBO J. 1995; 14: 2570-2579Crossref PubMed Scopus (348) Google Scholar). Circular dichroism studies, x-ray fiber diffraction and an analysis of the helical repeat of homopolymeric poly(dA·dT) tracts clearly showed that the homopolymer is structurally different from canonical B-DNA (31Wells R.D. Larson J.E. Grant R.C. Shortle B.E. Cantor C.R. J. Mol. Biol. 1970; 54: 465-497Crossref PubMed Scopus (501) Google Scholar, 32Arnott S. Selsing E. J. Mol. Biol. 1974; 88: 509-521Crossref PubMed Scopus (416) Google Scholar, 33Peck L.J. Wang J.C. Nature. 1981; 292: 375-378Crossref PubMed Scopus (246) Google Scholar). Failed attempts to assemble homopolymers into nucleosomes in vitro(34Rhodes D. Nucleic Acids Res. 1979; 6: 1805-1816Crossref PubMed Scopus (144) Google Scholar, 35Simpson R.T. Kunzler P. Nucleic Acids Res. 1979; 6: 1387-1415Crossref PubMed Scopus (157) Google Scholar) and the observation that cloned oligoadenosine regions were excluded from nucleosome formation (36Prunell A. EMBO J. 1982; 1: 173-179Crossref PubMed Scopus (90) Google Scholar) provided the basis for the view that poly(dA·dT) tracts are refractory to nucleosome assembly. More recent reports, however, present a different view on homopolymeric sequences function and structure. Herrera and Chaires (37Herrera J.E. Chaires J.B. Biochemistry. 1989; 28: 1993-2000Crossref PubMed Scopus (146) Google Scholar) showed that poly(dA·dT) tracts can assume a normal B-conformation, and other laboratories demonstrated that poly(dA·dT) sequences can be assembled into nucleosomes, in some cases even more favorably than heterogeneous-sequence DNA (38Losa R. Omari S. Thoma F. Nucleic Acids Res. 1990; 18: 3495-3502Crossref PubMed Scopus (68) Google Scholar, 39Hayes J.J. Bashkin J. Tullius T.D. Wolffe A.P. Biochemistry. 1991; 30: 8434-8440Crossref PubMed Scopus (64) Google Scholar, 40Puhl H.L. Gudibande S.R. Behe M.J. J. Mol. Biol. 1991; 222: 1149-1160Crossref PubMed Scopus (36) Google Scholar, 41Puhl H.L. Behe M.J. J. Mol. Biol. 1995; 245: 559-567Crossref PubMed Scopus (24) Google Scholar). We have shown that a poly(dA·dT) element present in the ILV1 promoter is required for efficient basal level expression and that the activity of the element is partially dependent on Dat1p, a poly(dA·dT) DNA-binding protein (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar). Analysis of ILV1 chromatin in the absence of Dat1p and in an ILV1-lacZ fusion containing a deletion of the downstream poly(dA·dT) element, showed no difference to a wild-type situation (data not shown), suggesting that theILV1 downstream poly(dA·dT) element was not responsible for the nucleosome exclusion observed in the promoter region. If the nuclease accessible site is caused by a strong setting preference of the nucleosomes positioned at the HS borders, we would expect that insertion of 74 bp of DNA into a previously 180-bp long stretch might allow the assembly of an additional nucleosome, thereby eliminating or splitting the nucleosome-free region. On the other hand, if a single cis-acting element present within the promoter region were responsible for the nucleosome-free region, then insertions would cause an asymmetric shift in the position of the HS. Insertion of spacing DNA in the center of the hypersensitive region (Fig. 8), clearly shows that the entire promoter structure remains unaltered, accommodating the inserted sequences within the hypersensitive site with a concomitant enlargement of the nucleosome-free region. The observed size increase suggested to us that two elements might be required to generate the hypersensitive region, such as the two A·T-rich tracts flanking the ILV1 Reb1p-binding site. However, simultaneous deletion of both A·T-rich tracts present in theILV1 promoter had no effect on the chromatin structure of the locus (Fig. 9), excluding the possibility that these elements are responsible for the nucleosomal exclusion. In summary, we have established the chromatin structure of theILV1 locus, with a positioned array of nucleosomes covering the entire gene and promoter, and a hypersensitive region comprising all known cis-acting sequences. We have shown that Reb1p does not visibly affect chromatin structure of the ILV1promoter even though the Reb1p-binding site is required for normal expression. This shows that Reb1p functions at ILV1 not through exclusion of nucleosomes but by some other mechanism of activation. We have also shown that the two poly(dA·dT) elements do not play an in vivo role in antagonizing nucleosome assembly in the ILV1 promoter, supporting our previous findings (2Moreira J.M.A. Remacle J.E. Kielland-Brandt M.C. Holmberg S. Mol. Gen. Genet. 1998; 258: 95-103Crossref PubMed Scopus (13) Google Scholar) and suggesting that binding of Dat1p, a poly(dA·dT)-binding factor, rather than chromatin modulation might be the function of these elements in the ILV1 promoter context. What is then responsible for the strong nuclease sensitive site observed in the ILV1 gene? Our results strongly suggest that the DNA sequence itself plays a dominant role in orchestrating the chromatin structure at the ILV1 promoter. The DNA contained within the hypersensitive region would be intrinsically refractory to nucleosome formation, thus creating a nucleosome-free zone. By the same token, the DNA adjacent to the hypersensitive site would dictate the positioning of the nucleosomes that is so characteristic of this promoter. This scenario would explain the resiliency of the chromatin organization of the ILV1 promoter to the loss of individual factor binding sites and to localized DNA insertions and deletions. Shen and Clark (42Shen C.-H. Clark D.J. J. Biol. Chem. 2001; 276: 35209-35216Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) recently reported that also at the yeastCUP1 gene DNA sequence is likely to play a very important role in positioning nucleosomes, which together with our data suggests a major role for DNA sequence in dictating nucleosome positioning and exclusion in vivo. We are grateful to B. J. Reardon and E. Winter for generously providing strains EWY1002c-1 and BRY1004–3. We gratefully acknowledge the support provided by the Plasmid Foundation during the stay of J. M. A. M. in the Hörz laboratory in München." @default.
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• W1987928196 title "Neither Reb1p nor Poly(dA·dT) Elements Are Responsible for the Highly Specific Chromatin Organization at the ILV1Promoter" @default.
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