INTRODUCTION

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The packaging of eukaryotic DNA into nucleosomes presents a barrier to cellular processes that require specific contacts with DNA. During transcription, sequence-specific DNA binding proteins and the basal transcription apparatus must recognize and bind to appropriate promoter elements. Biochemical and genetic analyses demonstrate that the packaging of DNA into nucleosomes inhibits its stable association with transcription factors (2, 29, 37, 57). A number of cellular activities capable of facilitating factor binding to chromatin have been identified (3). These activities are thought to function by directly modifying chromatin structure.

The Saccharomyces cerevisiae gene PHO5 is a well-characterized system with which to study regulated gene expression. PHO5 encodes a secreted acid phosphatase whose transcription is regulated in response to environmental phosphate levels (for a review, see reference 32). When phosphate is plentiful, PHO5 expression is repressed; when phosphate is limiting, PHO5 expression is induced. Activation of PHO5 transcription requires two transcription factors: Pho4, a basic helix-loop-helix protein, and Pho2, a homeodomain protein (58). In vitro, Pho2 enhances the binding of Pho4 to two regulatory sequences in the PHO5 promoter, UASp1 and UASp2 (4, 5).

When transcription of PHO5 is activated, its promoter undergoes a dramatic change in chromatin structure (for a review, see reference 51). When yeast cells are grown in high-phosphate medium, two pairs of positioned nucleosomes flank a DNase I-hypersensitive site, which contains UASp1 (Fig. 1, +P_i). UASp2 and the TATA box are packaged into nucleosomes 2 and 1, respectively. In vivo footprinting experiments indicate that Pho4 does not bind the PHO5 promoter under repressing conditions (57). When environmental phosphate is limiting, the positioned nucleosomes no longer protect the PHO5 promoter, and Pho4 binds to UASp1 and UASp2 (Fig. 1, P_i). In vivo footprinting of Pho2 at the PHO5 promoter has not been performed, but in vitro experiments indicate that Pho2 binds to this region in coordination with Pho4 (4, 5). The process by which the four positioned nucleosomes become undetectable and the PHO5 promoter is rendered sensitive to nucleases is termed the chromatin transition.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Chromatin structure of the PHO5 promoter from yeast grown in high- and low-phosphate media. Open circles, positioned nucleosomes; dark ovals, identified upstream activating sequences (UASs); T, location of the TATA box.

The mechanism by which PHO5 chromatin structure is changed during induction is unknown. However, a number of in vivo studies have provided some clues. The PHO5 chromatin transition is independent of transcription and DNA replication, as the loss of nucleosome positioning is unaffected by deletion of the PHO5 TATA box (18) and occurs when cell division is prevented (44). The Pho4 transcriptional activation domain is required for the PHO5 chromatin transition (52) but is not required for binding to naked DNA (19). If the activation domain is dispensable for binding to chromatin as well as to naked DNA, it may be required for interaction with a chromatin-remodeling activity or may be capable of changing chromatin structure itself.

It remains to be determined if factors besides Pho4 and Pho2 are required for PHO5 chromatin rearrangement. Several activities known to modify chromatin structure have been identified in yeast, and a few have been tested for a role in PHO5 induction. Loss-of-function mutations in several components of the ATP-dependent chromatin-remodeling complex Swi-Snf do not affect induction of acid phosphatase activity (10, 20, 45) or changes in PHO5 chromatin structure (20). PHO5 mRNA levels in high- and low-phosphate media are unaffected by mutations in the histone acetylase gene GCN5 (40), although gcn5 mutants have an unusual PHO5 promoter chromatin structure under partially inducing conditions (21). The PHO5 chromatin transition may involve the RNA polymerase holoenzyme, as artificial recruitment of the holoenzyme to PHO5 results in a promoter that is constitutively nuclease sensitive (20).

The reconstitution of chromatin rearrangement in vitro has allowed the isolation of several chromatin-remodeling activities from Drosophila embryo (22, 54, 56) and HeLa cell (35) extracts. It has been difficult, however, to obtain genetic evidence that these activities are involved in the transcriptional regulation of specific genes in vivo. In contrast, studies employing S. cerevisiae have a singular advantage in that this organism is easily manipulated in both biochemical and genetic experiments. This allows any result obtained in vitro to be rapidly tested for relevance in vivo. We describe here the reconstitution of the S. cerevisiae PHO5 chromatin transition in vitro. We propose to use this biochemical system to identify the components required for the chromatin rearrangement at the PHO5 promoter and to elucidate the mechanism by which it occurs.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmid construction. pTA-PHO5 was constructed in two steps. First, a BamHI-to-SpeI fragment from pACD5 (62), containing PHO5 sequence from nucleotide 542 to 1466, was inserted into pBluescript II KS to create pBSPHO5. Next, a 1.5-kb EcoRI fragment consisting of the TRP1/ARS1 locus was released from pTA-R (41) and inserted into the EcoRI site of pBSPHO5 such that PHO5 and TRP1 are transcribed in opposite directions, creating plasmid pTA-PHO5. To obtain pTA-p1p2, the hexanucleotide Pho4 binding sites at UASp1 (CACGTT) and UASp2 (CACGTG) in pTA-PHO5 were replaced precisely with a SpeI site (ACTAGT) and a BamHI site (GGATCC), respectively. In pTA-PHO5-ATG, the sequence AATGTT containing the translational start site was replaced with the sequence AGATCT, creating a BglII restriction site. Bacterial replication and selection sequences were removed from all minichromosome constructs by digestion with NotI, and the minichromosome circles were self-ligated before introduction into yeast. pRSPHO4 was constructed by inserting a BamHI-to-HindIII fragment of pACD4 (62) containing the PHO4 promoter and open reading frame into pRS426.

Strains. S. cerevisiae YS18 (47) was used in all experiments. Northern analysis was performed with strains EY0244 (wild type), EY0168 (pho3 pho5), and EY0168 harboring pTA-PHO5. For indirect end labeling, EY0255 (pho2 pho4 pho80) harboring pTA-PHO5 was used. For analysis of chromatin remodeling of episomal PHO5 in vivo, we used strains EY0246 (pho3 pho5 pho4 pho80) and EY0243 (pho3 pho5 pho80), harboring either pTA-PHO5-ATG or both pTA-PHO5-ATG and pRSPHO4. For minichromosome purification and nuclear extract preparation, either EY0255 or EY0579 (pho2 pho4 pho80 snf6) was used. To make EY0579, SNF6 was disrupted in a diploid by two-step gene replacement with pEY110 (16). After sporulation, snf6 haploid strains were identified by Southern blotting.

Northern blotting. Cell cultures were grown in medium lacking inorganic phosphate for 6 h as described previously (25), and total RNA was prepared as described previously (12). RNA was quantitated, and 20 µg of each sample was loaded on 6.7% formaldehyde-1.5% agarose gels and run in 1× E buffer (20 mM MOPS [morpholinepropanesulfonic acid] [pH 7.0], 5 mM Na acetate, 0.5 mM EDTA). The RNA was blotted to nylon and probed as described for Southern blotting below.

Preparation of PHO5 minichromosomes. PHO5 minichromosomes were prepared by the first steps of the procedure described by Simpson and colleagues (13, 42), with some modification. EY0255 or EY0579 cells harboring pTA-PHO5 or pTA-p1p2 were grown in 9 liters of synthetic medium to an A₆₀₀ of 1.0. Cells were pelleted, washed with water, and incubated at 30°C for 30 min in a freshly prepared solution of 0.7 M -mercaptoethanol-20 mM EDTA. The cells were washed once with 1 M sorbitol and resuspended in 500 ml of lyticase buffer (1.2 M sorbitol, 50 mM Tris-Cl [pH 8.0], 5 mM -mercaptoethanol). Spheroplasting was performed with 1 ml of crude recombinant lyticase (46) per g (wet weight) of cells for 30 min at 30°C. All subsequent manipulations were at 0 to 4°C. A swinging-bucket rotor (Sorvall HB-6) was used in all centrifugation steps unless otherwise noted. The spheroplast pellet was washed two times with 1 M sorbitol and then thoroughly resuspended in 240 ml of Ficoll buffer (18% Ficoll, 20 mM MOPS-NaOH [pH 6.8], 1 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride [PMSF]) and Dounce homogenized by hand (Wheaton 40-ml Dounce homogenizer), 10 times with the loose pestle and 5 times with the tight pestle. The lysate was layered over an equal volume of glycerol-Ficoll buffer (20% glycerol, 7% Ficoll, 20 mM MOPS-NaOH [pH 6.8], 1 mM MgCl₂, 1 mM PMSF) and spun at 11.5 krpm for 30 min. The pellet was resuspended in 20 ml of Ficoll buffer and centrifuged at 4.5 krpm for 15 min. The supernatant was transferred to a fresh chilled tube, and nuclei were collected by centrifugation at 11.5 krpm for 25 min. Pelleted nuclei were flash frozen at 80°C, thawed on ice, and incubated for 1 to 2 h in 9 ml of elution buffer {200 mM NaCl, 5 mM MgCl₂, 10 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]-NaOH [pH 7.3], 0.5 mM EGTA, 5 mM -mercaptoethanol, 1 mM PMSF}. Nuclei were pelleted by centrifugation at 11.5 krpm for 10 min, and the eluate was split between two 35-ml 0.4 to 1 M sucrose gradients made in elution buffer supplemented to a final NaCl concentration of 250 mM. The gradients were spun at 45 krpm for 80 min in a VTi50 rotor, braked to 10 krpm, and then allowed to coast to a stop. DNA was purified from 50 µl of each 1-ml fraction and assayed on ethidium bromide-stained agarose gels. Minichromosome-containing fractions were pooled, concentrated 10-fold on Centri-Prep concentrators that had been preblocked with insulin, and stored at 80°C in aliquots.

Southern blotting. Samples were loaded on 1.2% agarose gels and run in 0.5× Tris-borate-EDTA at 4 V per cm for 4 h. Gels were prepared as described previously (43) and blotted to nylon membranes (Amersham) overnight in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Prehybridization and hybridization with random-prime-labeled probes were performed in Rapid Hyb Buffer (Amersham). Typically, restriction fragments of 100 to 300 bp embedded in low-melting-point agarose were random prime labeled overnight at room temperature.

Micrococcal nuclease digestion and indirect end labeling of chromosomal or episomal PHO5 in vivo. Cells collected from 500 ml of cell culture grown to an A₆₀₀ of 1.0 were spheroplasted as in the minichromosome preparation, washed three times with 1 M sorbitol, and resuspended in 2 ml of digestion buffer A (1 M sorbitol, 50 mM NaCl, 10 mM Tris-Cl [pH 7.5], 5 mM MgCl₂, 1 mM CaCl₂, 1 mM -mercaptoethanol). Aliquots of 200 µl were placed in tubes containing 0.1 to 100 U of micrococcal nuclease (Worthington). Two hundred microliters of buffer B (buffer A plus 0.15% Nonidet P-40) was added to each tube, and the reaction mixtures were incubated at 37°C for 5 min. Reactions were stopped with 1/10 volume of 5% sodium dodecyl sulfate (SDS)-250 mM EDTA. DNA was purified by digestion with 200 µg of proteinase K per ml at 37°C for 2 h, followed by phenol-chloroform extraction, RNase treatment, and ethanol precipitation. For indirect end labeling of minichromosomes, 1/10 of each sample was digested with NgoMI or XmnI; genomic PHO5 DNA was digested with StuI. The Southern blot probes for analysis of episomal PHO5 and TRP1 nucleosome positioning were derived from an NgoMI-to-BglII fragment of the TRP1 gene and an XmnI-to-ScaI fragment of PHO5, respectively. As pTA-PHO5 is maintained in high copy, hybridization of this probe to the chromosomal TRP1 or PHO5 locus did not interfere with analyses of minichromosomal chromatin structure. For analysis of chromosomal PHO5, the probe was derived from a StuI-to-ApaI fragment of the PHO5 upstream region.

Chromatin remodeling in vivo. EY0246 and EY0243, harboring either pTA-PHO5-ATG or both pTA-PHO5-ATG and pRSPHO4, were cultured, spheroplasted, treated with micrococcal nuclease, and Southern blotted as described above. Probes for analysis of nucleosome 2 and nucleosome +1 correspond to ClaI-to-BstEII (probe A in Fig. 2) and DraI-to-SalI (probe B in Fig. 2) fragments of pBSPHO5, respectively. Southern blots were analyzed by phosphor screen autoradiography, and quantitative area analysis was performed with ImageQuant software.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Map of pTA-PHO5. Bacterial selection and replication sequences were removed, and a 3.5-kb circle containing PHO5 and the TRP1/ARS1 locus were religated to form pTA-PHO5. Black arrows, directions of transcription of PHO5 and TRP1; open circles, locations of three positioned nucleosomes on the PHO5 promoter; bars, sequences from which probes A and B were derived.

Preparation of recombinant Pho4. Escherichia coli BL21 harboring a T7-PHO4 expression vector (24) was grown in 3 liters of L broth supplemented with 50 µg of carbenicillin per ml to an A₆₀₀ of 0.4. IPTG (isopropyl--D-thiogalactopyranoside) was added to 0.4 mM, and the culture was grown for 2 h at 37°C. Cells were harvested, washed with B(0.1) (10% glycerol, 20 mM PIPES-NaOH [pH 7.3], 1 mM dithiothreitol, 1 mM PMSF, 1 µg of pepstatin A per ml, 0.1 M NaCl), and resuspended in 40 ml of B(0.1). Cells were lysed on ice by sonication and centrifuged at 16 krpm for 20 min in a Sorvall SS-34 rotor at 4°C. The lysate was treated with 10 U of DNase I, clarified through a 0.22-µm-pore-size filter, and loaded onto a 10-ml SP-Sepharose High Performance (Pharmacia) column. Pho4 was eluted with a linear gradient from 100 to 1,000 mM NaCl. Fractions containing Pho4 were pooled, adjusted to the conductivity of B(0.1) by dilution with B(0) [equivalent to B(0.1) except containing 0 M NaCl], loaded onto a BioScale S5 column (Bio-Rad), and eluted as before. This procedure yielded approximately 15 mg of Pho4, which appeared as a single band in SDS-polyacrylamide gel electrophoresis with Coomassie blue staining.

Preparation of S(0.3) extract. Nuclear extract was prepared from EY0255 or EY0579 as described previously (33), with the following modifications. Recombinant lyticase was used for spheroplasting, and lysis was performed with a hand-held glass Dounce homogenizer, as described above. The final protein pellet was resuspended in S(0.1) (20 mM HEPES-KOH [pH 7.9], 10% glycerol, 1 mM EDTA, 0.1 M K acetate, 1 µg of pepstatin A per ml, 1 mM PMSF). For fractionation, 5 mg of nuclear extract was applied to a 1-ml SP-Sepharose Fast Flow (Pharmacia) column, washed with S(0.1), and step eluted with S(0.3). [S(0.3) is equivalent to S(0.1) except that it contains 0.3 M K acetate.] Fractions containing protein were pooled and concentrated 10-fold with Centricon concentrators.

In vitro chromatin remodeling. Ten microliters of minichromosomes (approximately 10 ng of DNA in 250 mM NaCl-5 mM MgCl₂-10 mM PIPES-NaOH [pH 7.3]-0.5 mM EGTA) was incubated in 50-µl reaction mixtures containing 12 mM HEPES-NaOH (pH 7.5), 6 mM MgCl₂, 1 mM dithiothreitol, 5% glycerol, and 0.5 mM CaCl₂; 2 µg of poly(dC)-poly(dG) per ml and 0.1 mg of bovine serum albumin per ml were also added as nonspecific competitors. After addition of Pho4 (90 nM), Pho2 (approximately 20 nM), and 1 µg of S(0.3), the reaction mixtures were incubated at room temperature for 15 min. An ATP regeneration mix (final concentrations of 0.2 µg of creatine kinase per ml in 10 mM glycine [pH 8], 30 mM creatine phosphate, and 0.5 mM ATP or adenylyl imidi-diphosphate [AMP-PMP] was added, and reaction mixtures were incubated at 30°C for 30 min. The reaction mixtures were split and digested with either 0.1 or 0.05 U of micrococcal nuclease for 5 min at 37°C. Digestion was stopped with 1/10 volume of 5% SDS-250 mM EDTA. DNA was purified by overnight treatment with 200 µg of proteinase K per ml at 37°C, two phenol extractions, chloroform extraction, and ethanol precipitation. Samples were analyzed by Southern blotting as described above and probed as described for in vivo chromatin remodeling.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Episomal PHO5 is transcribed in response to phosphate starvation. Minichromosomes, or circular plasmids packaged into chromatin, have been purified from S. cerevisiae for the analyses of transcription (48), retroviral integration (41), centromere function (27), and chromatin structure (7, 53). We modified a 1.5-kb TRP1/ARS1 circle by inserting the PHO5 promoter and open reading frame to form pTA-PHO5 (Fig. 2). The PHO5 promoter fragment that we inserted includes sequence that is packaged into nucleosomes 1 through 3. This sequence is sufficient for phosphate-regulated transcription of PHO5 from a plasmid and for appropriate positioning of the three promoter nucleosomes (6, 18).

pho5

pho5

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Northern analysis of chromosomal and episomal PHO5 expression in response to phosphate starvation. A PHO5+ strain, a pho5 strain, and a pho5 strain harboring pTA-PHO5 were grown for 6 h in medium either containing (+) or lacking () inorganic phosphate (Pi). PHO5 transcript levels were quantified and normalized to the actin signal.

Episomal PHO5 has correctly positioned nucleosomes under repressing conditions. Micrococcal nuclease digestion followed by indirect end labeling is used as an assay for positioned nucleosomes in vivo (34, 61). We used this technique to compare the chromatin structure of chromosomal PHO5 with that of episomal PHO5 (Fig. 4). Spheroplasts with an intact chromosomal PHO5 locus harboring pTA-PHO5 were treated with micrococcal nuclease, and indirect end labeling was performed. Analysis of chromosomal PHO5 revealed four positioned nucleosomes (Fig. 4A, lane 3), which correspond to those mapped previously (1). Three similarly positioned nucleosomes were detected on the PHO5 promoter on pTA-PHO5 in vivo (compare lanes 3 and 7). The sequence upstream of nucleosome 3 on pTA-PHO5 (starting at the BamHI site) is the start of the TRP1 gene, which had a noticeably different pattern than the corresponding region of the chromosomal PHO5 gene.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   Nucleosomes on the PHO5 promoter have similar positions on the chromosome and on pTA-PHO5 and are not changed upon PHO5 minichromosome preparation. (A) Spheroplasts were treated with micrococcal nuclease (MNase), and the DNA was purified. For size standards, untreated DNA was digested with BamHI (lanes 1 and 5) or DraI (lanes 2 and 6). For analysis of chromosomal PHO5, samples were digested with StuI, Southern blotted, and hybridized to a probe derived from a StuI-to-ApaI fragment of the PHO5 upstream region. For analysis of episomal PHO5, samples were digested with NgoMI, and the probe used was derived from an NgoMI-to-BglII fragment from TRP1. (B) Partially purified PHO5 minichromosomes were treated with MNase, and the DNA was purified. Samples digested with NgoMI were probed with an NgoMI-to-BglII fragment from TRP1; samples digested with XmnI were probed with an XmnI-to-ScaI fragment from PHO5. The schematics show inferred locations of nucleosomes (open circles) on PHO5 and pTA-PHO5. The grey and black arrows represent PHO5 and TRP1 sequences, respectively. The location of the hypersensitive site HS2 on chromosomal PHO5 is indicated with an arrow.

pTA-PHO5 is remodeled in vivo. To test if the promoter chromatin of episomal PHO5 could be remodeled in vivo, we analyzed changes in chromatin structure by digestion with micrococcal nuclease followed by Southern blotting. This assay has been previously employed for analysis of the PHO5 chromatin transition (1, 44, 50). To analyze changes in chromatin structure at the PHO5 promoter, we used a probe derived from the PHO5 sequence packaged into nucleosome 2 (Fig. 2, probe A). A pattern of nucleosomal bands implies that PHO5 promoter sequence complementary to probe A is packaged into nucleosome 2 and is thereby protected from digestion. The disappearance of these bands implies that nucleosome 2 no longer protects the underlying DNA.

pho4

pho80

View larger version (26K): [in this window] [in a new window]   FIG. 5.   In vivo chromatin remodeling of episomal PHO5. Spheroplasts from the indicated strains were treated with micrococcal nuclease (MNase), and the DNA was purified and Southern blotted. (A) The blot was probed with probe A (Fig. 2). Data from each sample were quantified, and the distance from the top of the gel was graphed against the signal density. (B) The blot shown in panel A was stripped and reprobed with probe B (Fig. 2).

Preparation of PHO5 minichromosomes with intact chromatin structure. With evidence that the PHO5 promoter on pTA-PHO5 has correctly positioned nucleosomes, that its transcription is regulated by environmental phosphate levels, and that it can be remodeled in vivo, we developed a procedure to prepare PHO5 minichromosomes for in vitro study. Our protocol was based on the first steps of a purification procedure developed by Simpson and colleagues (13, 42). Our goals were to remove genomic DNA and cellular debris in a manner gentle enough to leave minichromosomal chromatin intact.

pho2 pho4

In vitro remodeling of PHO5 minichromosomes requires Pho4 and Pho2, hydrolyzable ATP, and a fraction of nuclear extract. The scheme of our in vitro chromatin-remodeling experiments is outlined in Fig. 6A. PHO5 minichromosomes were mixed with transcription factors and nuclear extract in a reaction mixture. A source of energy was then added, and the remodeling reaction was allowed to proceed. Reaction mixtures were split, digested with micrococcal nuclease, transferred to nylon, and probed with sequence corresponding to nucleosome 2 as for analysis of in vivo remodeling.

View larger version (35K): [in this window] [in a new window]   FIG. 6.   In vitro chromatin remodeling of PHO5 minichromosomes requires Pho2, Pho4, S(0.3), and ATP and is localized predominantly to the PHO5 promoter. (A) Schematic of the in vitro remodeling reaction. MC, minichromosome; MNase, micrococcal nuclease. (B) Reaction mixtures were assembled and incubated as shown in panel A and then split and digested with micrococcal nuclease. Samples were purified, electrophoresed, and transferred to nylon. Southern blotting was performed with probe A, and data were graphed as in Fig. 5. (C) The blot shown in panel B was stripped and reprobed with probe B.

View larger version (15K): [in this window] [in a new window]   FIG. 7.   Hydrolyzable ATP is required for in vitro chromatin remodeling of PHO5 minichromosomes. Either buffer (sample 1), ATP (sample 2), or AMP-PMP (sample 3) was included in chromatin-remodeling reactions with PHO5 minichromosomes, Pho4 and Pho2, and S(0.3). Samples were analyzed as described for Fig. 6. MNase, micrococcal nuclease.

S(0.3)-, ATP-, and Pho4-dependent chromatin remodeling is restricted to the PHO5 promoter region. In vivo, the loss of positioned nucleosomes in response to phosphate starvation is restricted to the four positioned nucleosomes on the PHO5 promoter (1). We tested if this was true of the remodeling observed in vitro by stripping and reprobing the Southern blot shown in Fig. 6B with a sequence underlying nucleosome +1. When analyzed with this probe, all samples produced a largely nucleosomal pattern (Fig. 6C). Thus, the dramatic change in chromatin structure observed in the presence of Pho4 and Pho2, S(0.3), and ATP at the PHO5 promoter does not extend significantly into the PHO5 open reading frame.

Pho4 can partially remodel PHO5 chromatin in the absence of Pho2. Overexpression of Pho4 can partially suppress the PHO5 expression defect of a pho2 strain (17). We therefore tested if Pho4 was capable of supporting chromatin remodeling of PHO5 minichromosomes in vitro without Pho2. As indicated in Fig. 8, Pho4 is capable of supporting partial remodeling of PHO5 minichromosomes in the presence of S(0.3) and ATP without Pho2 (sample 2). However, when Pho2 is included in the remodeling reaction mixture and Pho4 is left out, no remodeling is observed (sample 1). These results demonstrate that the transcription factor Pho4 is required for S(0.3)- and ATP-dependent chromatin remodeling of PHO5 minichromosomes.

View larger version (31K): [in this window] [in a new window]   FIG. 8.   In vitro chromatin remodeling of PHO5 minichromosomes in the absence of Pho2 requires Pho4, UASp1, and UASp2. Wild-type (WT) PHO5 minichromosomes were tested for chromatin remodeling in the presence of either Pho2 (sample 1) or Pho4 (sample 2). Minichromosomes lacking the Pho4 binding sites at UASp1 and UASp2 (p1p2) were purified and tested in parallel (samples 3 and 4). S(0.3) and ATP were included in all reaction mixtures. MNase, micrococcal nuclease.

Pho4-dependent chromatin remodeling requires UASp1 and UASp2. Deletion of a 26-bp region encompassing the Pho4 binding site in UASp1 has no effect on the assembly of repressive PHO5 chromatin structure, but it prevents Pho4 binding (57) and the chromatin transition (18) in vivo. These observations indicate that binding of Pho4 to the hypersensitive region is not required for nucleosome positioning yet is required for changes in chromatin structure upon induction. We wished to test if the Pho4-dependent remodeling observed in the absence of Pho2 requires specific binding by Pho4 to UASp1 and UASp2. A version of pTA-PHO5, pTA-p1p2, in which the two Pho4 binding sites in UASp1 and UASp2 were replaced precisely with restriction sites, was constructed. Wild-type and p1p2 minichromosomes were prepared in parallel and tested for remodeling in the in vitro system. Whereas the PHO5 promoter of wild-type minichromosomes was partially remodeled in the presence of Pho4 (Fig. 8, sample 2), no Pho4-dependent remodeling of p1p2 minichromosomes was observed (Fig. 8, sample 4). This result demonstrates that one or both of the Pho4 binding sites in UASp1 and UASp2 are required for Pho4-dependent chromatin remodeling of PHO5 minichromosomes in vitro.

PHO5 chromatin remodeling in vitro does not require the Swi-Snf complex. We reasoned that the factor(s) supplied by the S(0.3) extract might have ATPase activity, since ATP is required for in vitro remodeling. The yeast remodeling factor Swi-Snf contains an ATPase, which is required for its function (30). It was therefore possible that the putative ATP-dependent activity in S(0.3) was the Swi-Snf complex. As Swi-Snf is not required for PHO5 chromatin remodeling in vivo (10, 20, 45), it was of interest to determine if PHO5 minichromosome remodeling required this complex in vitro.

snf6

snf6

View larger version (36K): [in this window] [in a new window]   FIG. 9.   Swi-Snf is not required for in vitro chromatin remodeling of PHO5 minichromosomes. S(0.3) extract and minichromosomes were purified from a SNF6+ (samples 1 and 2) or snf6 (samples 3 and 4) strain and assayed for chromatin remodeling in the presence and absence of Pho2 and Pho4. An ATP regeneration mix was added to all samples. MNase, micrococcal nuclease.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have developed an in vitro system in which PHO5 minichromosomes undergo promoter chromatin remodeling. To obtain chromatin templates with positioned nucleosomes, an important feature of the repressed PHO5 promoter in vivo, we purified minichromosomes carrying the PHO5 gene from yeast. Chromatin remodeling in our system required the presence of the transcriptional activators Pho2 and Pho4, a fraction of S. cerevisiae nuclear extract, and hydrolyzable ATP.

In vitro chromatin remodeling of PHO5 minichromosomes was localized to the PHO5 promoter and did not require the Swi-Snf complex. Specific binding by Pho4 to UASp1 and UASp2 was required for Pho4-dependent chromatin remodeling. As these are also characteristics of the PHO5 chromatin transition in vivo, we believe that this system will allow the identification of physiologically relevant chromatin-remodeling activities.

Choice of chromatin template. The chromatin structures of the PHO5 promoter under repressing and inducing conditions have been characterized (51). In high-phosphate medium, four nucleosomes are positioned on the PHO5 promoter such that UASp1 is in a hypersensitive region between nucleosomes 2 and 3, UASp2 is packaged into nucleosome 2, and the TATA box is packaged into nucleosome 1. These four nucleosomes lose their positioning upon PHO5 induction and no longer protect the promoter from nuclease digestion (Fig. 1).

Definition of chromatin remodeling. To describe and characterize our in vitro system, we have defined chromatin remodeling of PHO5 minichromosomes as an increase in micrococcal nuclease sensitivity at the DNA sequence packaged into nucleosome 2. Samples probed with sequence from nucleosome 2 (Fig. 6B) showed the same loss of nucleosomal bands as when they were probed with sequence from nucleosome 3 or sequence from all three nucleosomes (data not shown). In contrast, samples probed with sequence from nucleosome +1 (Fig. 6C) or +5 (data not shown) were mostly nucleosomal. Remodeling as defined here thus extends from nucleosome 1 to nucleosome 3, but it does not extend into the PHO5 open reading frame.

Role of Pho4 in PHO5 chromatin remodeling. The activation domain of Pho4 is required for the PHO5 chromatin transition in vivo. A version of Pho4 that lacks the activation domain is capable of binding to UASp1, but not to UASp2, under inducing conditions in vivo (52). When this Pho4 mutant is expressed in place of full-length Pho4, the PHO5 promoter remains packaged into positioned nucleosomes, and there is no expression of PHO5 under inducing conditions.

Identity of the remodeling activity in the S(0.3) extract. The activity contained in the S(0.3) extract may be a member of the rapidly growing family of ATP-dependent chromatin-remodeling machines, each of which contains a member of the Snf2 family of helicase-like ATPases (15). These complexes are capable of altering the DNase I digestion pattern of a mononucleosome, facilitating factor binding to sites within nucleosomes, and potentiating activation of transcription from chromatin templates (8). Members of this family thus appear to have in common the ability to modify histone-DNA contacts in an ATP-dependent manner.

Reconstitution of the PHO5 chromatin transition. We describe here the reconstitution of PHO5 chromatin remodeling in vitro. The PHO5 chromatin transition was studied in S. cerevisiae, a model organism that is amenable to both genetic and biochemical experiments. This allowed us to compare the in vivo PHO5 chromatin transition with our in vitro remodeling reaction, and it provides a way to confirm the physiological relevance of future in vitro results.