Vol. 11, No. 10
MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 5301-5311 0270-7306/91/105301-11$02.00/0 Copyright C 1991, American Society for Microbiology
The Chromatin Structure of Saccharomyces cerevisiae Autonomously Replicating Sequences Changes during the Cell Division Cycle JULIE A. BROWN,t SCOTT G. HOLMES, AND M. MITCHELL SMITH* Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908 The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARSI is present near the TRP1 gene. Cells blocked in G, either by a-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.
may include bent DNA (58, 74), transcription termination (59), multiple sequences homologous to the core consensus (24, 45), a low free energy of unwinding (69, 70), or a favorable chromatin conformation (3, 53). Chromatin structure may play an important role in the function of replication origins. Several origins have been shown to be in a chromatin structure accessible to DNA endonucleases. For example, the replication origins of Tetrahymena ribosomal DNA (44) and the origin of the simian virus 40 minichromosome are maintained in nucleosome-free regions (31, 51) and the DNase I sensitivity of the simian virus 40 replication origin is greater in postreplicative than in prereplicative minichromosomes (10). Yeast plasmid ARS elements are also located in nucleosome-free chromatin structures. The DNAs of the TRPI-ARSJ circle (39, 67, 68) and the 2,um plasmid (15, 36, 73) are assembled with nucleosomes in vivo to form minichromosomes; however, the ARS elements in both of these plasmids are located in nucleosome-free regions. The positioning of a nucleosome within ARSI is sufficient to inactivate its replication function
Autonomously replicating sequence (ARS) elements of the yeast Saccharomyces cerevisiae were first identified by their ability to enable plasmids to replicate as independent episomal minichromosomes (26, 64, 65). Considerable evidence has accumulated to suggest that ARS elements are origins of DNA replication (reviewed in references 42 and 71). For example, the replication of ARS plasmids take places only during S phase, is under the same genetic control as chromosomal replication, and occurs only once per division cycle (16, 77, 78). Electron microscopy of the 2 ,Lm plasmid (43) and the ribosomal DNA locus (50) showed that at least some of the mapped replication bubbles were coincident with known ARS elements. Recently, in vivo replication origins have been localized by two-dimensional gel mapping techniques to the 2,um plasmid ARS (6, 27), ARSI on a plasmid (6), the chromosomal ribosomal DNA ARS (35), an ARS on chromosome III called A6C (28), and ARSI on the chromosome (18). A detailed molecular examination of at least five different ARS elements has led to a consistent picture of the DNA sequence requirements for function (3, 5, 7, 9, 32, 33, 45, 62). The ARS element is a bipartite structure composed of an 11-bp core consensus sequence and a 3' flanking domain of approximately 60 bp. Distal sequences are not required for replication activity, although they may influence efficiency depending on the particular ARS. The core consensus was first detected by comparative sequence analysis (7, 63), and its importance has since been rigorously demonstrated by point mutations, deletions, and linker-scanning mutations. The requirement for a 3' flanking domain has been established by deletion analysis; however, unlike the core consensus, the 3' flanking domain does not depend on an absolute primary DNA sequence (3, 45). The required features of the 3' flanking domain are not completely known but *
In this report, we present the results of an examination of the chromatin structures of two ARS elements, the H4 ARS and ARSI, during the cell division cycle. These experiments were designed to address several aspects of ARS structure and function. First, we have sought to determine whether ARS elements preserve a specialized chromatin structure at their native chromosomal locations. Second, we have used synchronous cultures to investigate whether changes in ARS chromatin structure can be detected during the cell division cycle. Finally, we have investigated whether the changes in ARS structure depend on DNA replication. MATERIALS AND METHODS Strains and plasmids. Chromatin structure was analyzed in strains MSY159 (MATa ura3-52 ade2-101 lys2-801) and RM14-3A (MATa barn his6 cdc7-1 leu2-3 leu2-112 ura3-52 trpl-287). S288C (MATTa gal2 mal) was used for a-factor
t Present address: National Institutes of Health, Bethesda, MD 20892. 5301
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block by shifting the culture back to 23°C. Chromatin was made 20 to 40 min following the shift to the permissive
probel 1 kb
B A C
probe 2 0.5 kb
FIG. 1. Organization of the copy-I histone H3-H4 and the TRPI chromosomal loci. (A) Positions of the SMTI gene, the histone H3 and H4 genes, and the H4-associated ARS, shown relative to the position of the 6.7-kb HindIII fragment used in these studies. The H4 ARS core consensus sequence is positioned at the left of the box, with the T-rich strand of the consensus oriented 5' to 3'. The direction of gene transcription is shown by arrows. The location of the BamHI-HindIII fragment (probe 1) used for indirect end labeling is shown by the solid box. (B) Organization of the TRPI-ARSI chromosomal locus. The locations of the TRPI gene and the three ARS domains, A, B, and C, are shown relative to the position of the EcoRI fragment. The ARSI core consensus sequence (domain A) is shown as the solid bar, and the T-rich strand of the consensus is oriented 3' to 5'. The dotted line indicates the minimum extent of the B domain. The location of the EcoRI-RsaI fragment (probe 2) used for the indirect end-label mapping is shown by the solid box. Restriction enzyme sites: B, BamHI; E, EcoRI; H, HindIll; R, RsaI.
production. Plasmid pMS191 has been described previously (56). pJAB1 is a derivative of pBR322 containing the 1-kb BamHI-HindIII fragment from pMSl91. Plasmid YRp7 has been described previously (64). The structures of the Sc191 fragment and the TRPI-ARSI fragment are shown in Fig. 1. Synchronized cultures. Cells were blocked in G1 phase with a-factor as described by Hereford and Hartwell (22), using ot-factor produced as described by Hereford and Osley (23). Additional synthetic a-factor (Sigma) was added to the cultures at a concentration of 0.05 ,ug/ml to augment the natural ot-factor. For synchronous cultures, the at-factor was removed by filtration, and cells were washed with 1 to 2 volumes of water and then resuspended in 500 ml of MV medium (23) prewarmed to 28°C. Cells were then harvested for analysis at various times after release from at-factor. Synchrony was assessed by flow microfluorometry and fluorescence microscopic examination as described below. Cells arrested in G1 by nitrogen starvation were prepared by transferring exponentially growing cultures to a medium containing glucose and yeast nitrogen base without ammonium sulfate. Cells were incubated in this nitrogen starvation medium for 6 h, at which point approximately 90% of the cells were unbudded. Synchronized cultures of cdc7 cells were prepared by switching exponentially growing populations of cells from the permissive temperature (23°C) to the nonpermissive temperature (36°C). Incubation was continued at 36°C for 2 to 2.5 h until 70 to 80% of the cells displayed large buds. Cells were released from the cdc7
temperature. Flow microfluorometry and fluorescence microscopy. Cells for flow cytometry were fixed and stained with propidium iodide (11), and histograms were collected in an EPICS V fluorescence-activated cell sorter (Coulter Electronics, Inc.)
(57). Haploid cells arrested with a-factor, haploid cells blocked with nocodazole (30), and diploid cells blocked with nocodazole were used as standards for 1C, 2C, and 4C amounts of DNA per cell. For the analysis of synchronous cultures, the samples from each time point were seeded with a constant number of cells containing a 4C amount of DNA to provide an internal control for fluorescent staining. Cells for fluorescence microscopy were fixed with methanol-acetic acid (3:1), stained with 4,6-diamidino-2-phenylindole (Boehringer Mannheim), and examined on a fluorescence microscope at 420 nm (75). Approximately 200 cells were counted and scored as either an unbudded cell, a budded cell with a single nucleus, or a budded cell with two nuclei. Mapping of DNase I HS sites. Synchronized yeast cultures (2 x 109 to 6 x 109 cells) were harvested by filtration and washed with 2 volumes of water. Nuclei were isolated essentially according to Lohr and Ide (37) except that preincubation was omitted, cells were incubated at 37°C for S to 15 min with Lyticase (20 to 40 ,ug/ml; Sigma), and disrupted cells were centrifuged at low speed (3,000 x g) only once. DNase I treatment and DNA purification were done as described by Bloom and Carbon (2) except that the DNase I concentration was varied from 1 to 16 ,ug/ml, digestions were carried out for 5 min, and RNase A treatment was omitted. The DNA was dissolved in an appropriate restriction endonuclease buffer plus 4 mM spermidine and incubated overnight with restriction enzyme. DNA from approximately 4 x 108 cells was fractionated on 1.3% agarose gels, along with DNA size markers consisting of pMS191 digested to completion with HindIll, and partially digested with either HpaII or HhaI. Transfer of the DNA to Nytran (Schleicher & Schuell) was performed as described by Southern (60). The DNase I hypersensitive (HS) sites were mapped by the technique of indirect end labeling (40, 76). The copy-I histone locus was mapped by using the BamHI-HindIII fragment from pJAB1 (Fig. 1, probe 1), and ARSI was mapped by using the 360-bp EcoRI-RsaI fragment from YRp7 (Fig. 1, probe 2). Fragments were labeled by random primer extension (17). The membranes were prehybridized in 50% formamide-10% dextran sulfate-1 M sodium chloride-1% sodium dodecyl sulfate (SDS)-100 ,ug of denatured herring sperm DNA per ml at 42°C for 2 to 4 h. Herring sperm DNA (for a final concentration of 200 jLg/ml) and the radiolabeled probe were added to the prehybridization buffer and incubated at 42°C overnight. Membranes were washed with a final stringency of 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% SDS at 65°C. Kodak XRP film was exposed to the membranes at -70°C for 1 to 14 days, using DuPont Cronex Hi-Plus intensifying screens. Autoradiographs were scanned on a Visage 2000 video densitometer. Data analysis. The correlation of the observed H4 ARS chromatin structure changes with respect to the timing of the cell division cycle was deduced from an analysis of the synchronous culture fractions. The ratios of the two DNase I HS peaks in the H4 ARS were used as a quantitative measure of the chromatin structure. The measured ratio, R, is given by
ARS CHROMATIN STRUCTURE
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R= fg(x)flx) x= o
where x is the position in the cell division cycle (from 0 to 1), g(x) is the division cycle age distribution for the cells in the sample and f(x) describes the ratio of peaks with respect to the position in the division cycle. The distribution of cell division cycle ages in each of the synchronous culture samples was estimated from the flow cytometry and morphological scoring data shown in Fig. 3 as described by Fraser and Barnes (19). A simple two-state model was used for the function f(x). The ARS chromatin was assumed to have a state A characterized by a starting ratio of DNase I HS site intensities. At some point in the division cycle, the chromatin structure was assumed to change to state B with a different ratio of site intensities, finally returning to state A later in the cycle. There are thus four variables in the model: (i) the starting ratio in state A, (ii) the time when the chromatin enters state B, (iii) the ratio of DNase I HS site intensities in state B, and (iv) the time when the chromatin returns to state A. By using the ratios measured from the DNase I HS site mapping data, the equation for R was solved for these four variables by the downhill simplex method of Nelder and Mead (41), using subroutines of Press et al. (47). RESULTS Chromatin structure of the H4 ARS. The H4 ARS, located 293 bp 3' of the H4 coding region, is composed of a core consensus element and a required 3' flanking domain (3, 25). The structure of a 6.7-kb HindlIl fragment containing the ARS is shown diagrammatically in Fig. 1 (56). This HindlIl fragment contains, in addition to the ARS element, three genes: SMTJ, the gene for inorganic pyrophosphatase (now called PPA) (34); HHTJ, the copy-I histone H3 gene; and HHFJ, the copy-I histone H4 gene. The chromatin structure of this locus was characterized by mapping the location of DNase I HS sites as described in Materials and Methods. As illustrated in Fig. 2A, distinct DNase I HS sites were present near the ends of the genes while the coding regions were relatively inaccessible to DNase I, an overall pattern similar to that found for many other genes (14, 20). HS sites were observed 5' of the SMTJ gene, between the 3' ends of SMTJ and H3, in the promoter region of H3 and H4, and 3' of the H4 gene. Distinct HS sites were also found at the H4 ARS element (arrowheads in Fig. 2A). In general, the HS region began near the 5' end of the core consensus element and spanned the minimal ARS sequences (25); the local pattern of DNase I HS sites at the ARS showed specific variations during the division cycle (see below). Thus, the H4 ARS element is in a chromatin structure that is hypersensiti'.' to DNase I digestion and presumably free of positioned . ucleosomes. To control for DNase I sequence prefei nces, purified DNA was used in the nuclease assay instead of nuclei. Figure 2B shows a comparison of the DNase I pattern of purified DNA (lane 1) and chromatin (lane 2). The DNA was more uniformly sensitive to nuclease, and most of those HS sites present did not correspond to HS sites in chromatin. The coding regions were more protected in chromatin than in purified DNA, whereas the H3-H4 intergene region and the ARS region of purified DNA appeared less sensitive than they were in chromatin.
7 8 9
InI? LI *
4. w w
4 j I.0 30.
FIG. 2. DNase I HS sites at the copy-I histone H3-H4 locus. (A) Results of indirect end-label mapping of DNase I HS sites at the copy-I histone locus. Cells were harvested at different times during synchronous culture, and isolated nuclei were partially digested with DNase I as described in Materials and Methods. DNA samples purified from nuclease-treated nuclei were digested to completion with HindlIl, fractionated on a 30-cm-long 1.3% agarose gel, transferred to a Nytran membrane, and hybridized with radioactively labeled probe 1 (Fig. 1A). The two HS sites near the ARS are indicated by arrowheads. Lanes: 1 to 3, cells arrested in G1 with a-factor; 4 to 6, cells taken 50 min after release from a-factor; 7 to 9, cells taken 75 min after release. In each set of three lanes, the concentrations of DNase I used for digestion were, from left to right, 2, 4, and 8 ,ug/ml. (B) Control DNase I digestion of naked DNA (lane 1) run next to a digestion of whe 'e nuclei isolated from an asynchronous culture (lane 2). The naked DNA was treated with 40 ng of DNase I per ml, and the nuclei were digested with 4 ,ug of DNase I per ml.
Cell cycle and synchronous cultures. The cell cycle of MSY159 was first characterized in exponentially growing cultures to determine the timing of the G1, S, and G2+M phases. The results of control experiments to calibrate the flow cytometry measurements are shown in Fig. 3A, trace 3. Haploid cells blocked prior to DNA replication with a-factor provided a control histogram for 1C DNA content, haploid cells blocked after DNA replication with nocodazole (a microtubule inhibitor ) provided a control histogram for 2C DNA content, and diploid cells blocked with nocodazole provided a control histogram for cells with a 4C DNA content. These populations showed three distinct fluorescence intensity peaks for the three different DNA contents. DNA histograms for cells from an exponentially growing culture of MSY159 are shown in Fig. 3A, traces 1 and 2; in trace 2, the sample was seeded with diploid cells blocked with nocodazole to provide an internal 4C DNA standard. As expected, the MSY159 cell population gave two peaks, corresponding to cells in the G1 (IC DNA content) and G2+M (2C DNA content) phases, and a distribution between these two peaks corresponding to cells in S phase. The lengths of the cell division cycle periods for MSY159 were calculated from the generation time of 95 min and the flow cytometry histograms as described previously (54, 57). The G1 phase was estimated to be 19 + 2 min, the S phase was 27 ± 9 min, and the G2+M phase was 49 + 5 min. Next we characterized the synchrony of MSY159 cultures that wou-d be used to prepare chromatin at different points in
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