ABSTRACT
The Snf-Swi complex of the yeast Saccharomyces cerevisiae has been shown to control gene expression by controlling chromatin structure. We have analyzed the promoter of the SUC2 gene, a gene strongly controlled by Snf-Swi, by a high resolution analysis of micrococcal nuclease digests. This analysis suggests that there are at least four nucleosomes positioned over the SUC2 TATA and UAS regions under conditions repressing SUC2 transcription. Under derepressing conditions this entire promoter region is much more sensitive to MNase digestion. Analysis of an snf2[Delta] mutant demonstrates that even under derepressing conditions the SUC2 promoter is resistant to MNase digestion. Thus, the Snf-Swi complex appears to control chromatin structure over both the SUC2 TATA and UAS regions. The presence of nucleosomes over both promoter regions may explain the strong requirement of SUC2 for Snf-Swi function.
Genetic, biochemical and molecular experiments have strongly suggested that the Snf-Swi complex controls eukaryotic gene expression by antagonizing chromatin-mediated repression of transcription (1 ). This multiprotein complex is strongly required for transcription of SUC2, a Saccharomyces cerevisiae gene encoding the enzyme invertase, required for growth of yeast cells on sucrose. The SUC2 gene is controlled by glucose repression: in the presence of high glucose SUC2 is transcriptionally repressed; in low glucose SUC2 is derepressed and transcription is increased >100-fold (2 ). Previous work has shown that under derepressing conditions snf/swi mutants have an ~10-fold lower level of SUC2 mRNA. Analysis of SUC2 chromatin structure in wild-type, snf2 and snf5 strains has shown that Snf-Swi functions by altering chromatin structure to a transcriptionally permissive conformation (3 ).
In order to gain a better understanding of the role Snf-Swi plays in regulation of chromatin structure at the SUC2 gene, we analyzed the SUC2 promoter chromatin structure by a high resolution analysis of digestion by micrococcal nuclease (MNase). Our results provide additional evidence for the presence of two nucleosomes previously identified over the TATA box and upstream of the TATA box. We have extended our knowledge of SUC2 promoter chromatin structure by demonstrating the presence of two additional nucleosomes positioned over the UAS region. Alterations in chromatin structure occur over this entire region upon derepression in wild-type cells. In snf2 mutants, in which Snf-Swi is not present, these changes in chromatin structure are not observed. Thus, Snf-Swi appears to be required for controlling at least four nucleosomes that cover the SUC2 TATA and UAS regions.
The two S.cerevisiae strains used in these studies are congenic to S288C except that they are GAL2+ (4 ). The two strains are FY120 (MATa his4-912[delta] lys2-128[delta] leu2[Delta]1 ura3-52) and FY458 (MATa his4-912[delta] lys2-128[delta] snf2[Delta]1::HIS3 his3[Delta]200 ura3-52). The snf2[Delta]1::HIS3 null allele has been previously described (5 ).
All media were prepared as described (6 ). YEP + 0.05% glucose was identical to YPD except that it contained 0.05% glucose instead of 2% glucose.
Glucose-repressed cultures were grown in YPD at 30oC and harvested at 2 × 107 cells/ml. For derepressed cultures, cells were grown in YPD to 2 × 107 cells/ml and then washed twice with water, then grown in an equal volume of YEP + 0.05% for 165 min at 30oC.
MNase was purchased from Boehringer Mannheim Biochemicals and stored at 6000 U/ml at -20oC in 50% glycerol, 2 mM CaCl2 and 50 mM Tris, pH 9.0. Taq polymerase was purchased from Promega.
From each culture, 4 × 109 cells were spun down and resuspended in 10 ml room temperature S buffer (1.1 M sorbitol, 20 mM KPO4, pH 7.0, 0.5 mM CaCl2) and 10 mM DTT (added fresh) and incubated for 15 min at 30oC. Cells were then centrifuged and all liquid was removed by aspiration. The cell pellet was resuspended in a total volume of 1 ml S buffer. To each 1 ml cells was added 1 ml S buffer with freshly added zymolyase T100 (1 mg/ml). Cells were shaken at 90 r.p.m. for 10-15 min at 30oC. Greater than 90% of the cells were converted to spheroplasts as monitored by lysis in a 50-fold excess of water. Spheroplasts were washed twice by adding 10 ml S buffer and centrifuged in a Sorvall H6000A rotor for 6 min at 1200 r.p.m. and resuspended gently.
For MNase digestion each sample was gently resuspended with a large bore pipet tip to a final volume of 600 µl in ice-cold buffer A (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 1 mM [beta]-mercaptoethanol and 0.5 mM spermidine). To begin digestion, 200 µl aliquots of the cell slurry were added to 200 µl prewarmed (37oC) buffer A with 0.15% NP40 and MNase (0.5, 1.5 or 5 U/ml final concentration) and incubated for 5 min at 37oC. The reactions were terminated by addition of 40 µl stop buffer (250 mM EDTA, 5% SDS) and incubated for an additional 15 min at 37oC.
To isolate the DNA, 160 µl 5 M KOAc were added to each digest and incubated on ice for 30 min. After spinning down the precipitate for 15 min in a microfuge, the supernatant was transferred to a new 1.5 ml microcentrifuge tube and the DNA precipitated by adding an equal volume of isopropanol. The DNA was pelleted by spinning in a microfuge for 10 min. The DNA pellet was then resuspended in 500 µl TE buffer with 100 µg/ml RNase A and incubated for 30 min at 37oC. DNA was extracted with an equal volume of phenol/chloroform. The aqueous phase was transferred to a fresh tube and DNA was precipitated by adding 67 µl 7.5 M NH4OAc and 500 µl isopropanol.
The naked DNA was prepared by starting with 8 × 109 cells. These cells were spheroplasted as above and resuspended in a final volume of 1200 µl buffer A and divided into 400 µl aliquots. Cells were lysed by adding 80 µl stop buffer to each tube. Then, 320 µl KOAc were added. RNase digests were then carried out as for the other samples. To digest with MNase, the DNA was resuspended in 300 µl buffer A containing 0.075% NP40. To start the digest, 100 µl buffer A + 0.075% NP40 containing MNase (0.01, 0.05 or 0.15 U/ml final concentration) were added. DNA was incubated for 1 min at 37oC. Digestions were stopped by addition of 10 µl 500 mM EDTA. Samples were then extracted with an equal volume of phenol/chloroform and precipitated with 3 M NaOAc.
All samples were resuspended in 100 µl water and 2 µl was run on a 1% agarose gel to determine the extent of digestion. For optimal analysis by primer extension methods the bulk of the DNA should run as a high molecular weight band. With increasing amounts of digestion a faint smear of DNA should be visible in the lane. If a nucleosome ladder is present, this indicates overdigestion.
In preparation for PCR analysis, DNA was digested with EcoRI (EcoRI cuts outside the SUC2 region undergoing PCR analysis). Following digestion the DNA was phenol/chloroform extracted and precipitated and resuspended in 50 µl H2O.
For primer extension analysis the following oligonucleotides were used. All oligonucleotides were purified by polyacrylamide gel electrophoresis before use. The annealing temperatures for each oligonucleotide are given in parentheses.
S13, 5'-gcacgGtgagctgtcgaagg-3' (63oC) [-755 to -736];
S11, 5'-GGTATGGTACGTTAGAAAGGC-3' (57oC) [-587 to -567];
SUC2-2, 5'-GGTACGCCCGATGTTTGCCTATTACC-3' (67oC) [-313 to -288];
SUC2-1, 5'-GGTAATAGGCAAACATCGGGCGTACC-3' (67oC) [-288 to -313];
SUC2-3, 5'-GTGAAGTGGACCAAAGGTCTATCG-3' (63oC) [+100 to +76].
Oligonucleotides (100 ng) were end-labeled with [[gamma]-32P]ATP and T4 polynucleotide kinase (7 ).
Primer extension analysis by PCR of DNA digested by MNase was carried out as described previously (8 ) except that 5 µg genomic DNA were used in each reaction and the following Taq buffer was substituted: 50 mM KCl, 10 mM Tris, pH 8.5, 2.5 mM MgCl2, 170 µg/ml BSA.
Following PCR and precipitation, DNA fragments were separated on an 8% sequencing gel and visualized by autoradiography.
Previous analysis of SUC2 promoter chromatin structure by indirect end-labeling suggested that in either repressed cells or in snf/swi mutants the presence of two positioned nucleosomes, one centered over the TATA box and the second located in between the TATA box and UAS (3 ,9 ,10 ). This analysis further suggested that in wild-type derepressed cells these nucleosomes were either absent or altered in such a way as to allow digestion of the DNA by MNase. Given the nature of indirect end-labeling experiments, the previous analysis did not allow accurate mapping of the MNase cleavage sites. In addition, we were unable to determine if MNase digestion was affected over the UAS region. Therefore, we set out to analyze the entire SUC2 promoter region from near the transcription initiation site (-40 relative to the ATG) upstream through the UAS region (to -650) by a high resolution analysis of MNase cleavage.
To analyze the position of the nucleosome over the SUC2 TATA box we used primer SUC2-2 and examined MNase cleavage under four sets of conditions: SNF2+ repressed and derepressed and snf2[Delta] repressed and derepressed. In SNF2+ repressed cells most of the DNA at the TATA box is not accessible to MNase digestion (Fig. 1 ). Two clusters of MNase cleavage sites are observed on the antisense strand; there is a doublet located at nucleotides -74 and -75 and a second cluster, composed of a triplet, located at nucleotides -215 to -217. The distance between the first cluster located at -74,-75 and the center of the second cluster is 146 bp, which corresponds to the length of DNA in a nucleosome. The TATA box, located at -133, is situated in the middle of the protected DNA region. When a SNF2+ strain is grown under derepressing conditions, a significant increase in DNA cleavage by MNase is observed, indicating an increase in accessibility. The increase in DNA accessibility observed in derepressed cells indicates that this nucleosome is altered when SUC2 is transcriptionally active. Similar results were obtained on the sense strand, although the observed cleavages were not as pronounced (data not shown). These results indicate that a nucleosome is positioned directly over the TATA box in repressed cells.
To determine if nucleosomes might also be positioned over the SUC2 UAS we examined the MNase cleavage pattern of the region upstream of the transcription initiation site to ~650 bp upstream of the initiation site using two different primers. This analysis was also done under the same four different conditions. First, using primer S11, we analyzed the antisense strand of the UAS. In chromatin from wild-type cells grown under repressing conditions we observed a large protected region of DNA extending from nucleotide -226 to a doublet located at nucleotides -490/-500 (Fig. 2 ). The length of the protected DNA corresponds to the amount of DNA in two nucleosomes and thus indicates the presence of two more nucleosomes upstream of the one over the TATA box. The chromatin from wild-type cells grown under derepressing conditions shows much more cleavage by MNase, again suggesting the absence or alteration of nucleosomes. Similar results were obtained with our analysis of the sense strand over the UAS region (data not shown). Using primer SUC2-1 to look further upstream we detected protection of a region ~150 bp in size (as determined by DNA size markers) that begins at the doublet located at -490/-500 and extends upstream to approximately -650(Fig. 3 ), indicating the presence of a fourth nucleosome. These results suggest that under glucose repressing conditions at least four nucleosomes are positioned over the SUC2 promoter, extending from the transcription initiation site back over the UAS. Furthermore, under derepressing conditions these nucleosomes become altered or removed to allow cleavage by MNase.
In this work we have examined the chromatin structure of the SUC2 promoter region by MNase digestion under both repressing and derepressing conditions and in both SNF2+ and snf2[Delta] genetic backgrounds. This analysis extends previous work in that it has examined a larger region of the promoter and at higher resolution than in past studies. Our results have produced two main findings. First, in a wild-type background SUC2 promoter chromatin is generally inaccessible to MNase when cells are grown under repressing conditions (high glucose) and it is very accessible to MNase when cells are grown under derepressing conditions. Second, in an snf2[Delta] background SUC2 promoter chromatin is inaccessible to MNase under both repressing and derepressing conditions. The MNase digestion pattern under repressing conditions suggests the presence of at least four nucleosomes over the SUC2 promoter, including the TATA box and the UAS (summarized in Fig 4 ). Because changes in chromatin structure were not observed in snf2[Delta] mutants, we conclude that Snf2 and the Snf-Swi complex are required for the alteration in chromatin structure at all four nucleosomes. While this work was in progress Gavin et al. (11 )also described studies of the chromatin structure at SUC2. Their experiments demonstrated positioned nucleosomes over a larger region of SUC2 than we examined in our studies. In addition, their studies demonstrate that a swi1 mutation, like a snf2 mutation, causes a repressing chromatin state, even under derepressing conditions.
We thank Igor Gavin and Bob Simpson for sharing unpublished results, and Sharon Roth for advice on the MNase digestion experiments. L.W. was supported by a post-doctoral fellowship from the American Cancer Society. This work was supported by NIH grant GM32967.
REFERENCES