Chromatin structure of transposon Tn903 cloned into a yeast plasmid

PLASMID

22, 143-150 (1989)

Chromatin Structure of Transposon

Tn903 Cloned into a Yeast Plasmid

FRANCISCO ESTRUCH, Jo& E. PfiREZ-ORTfN, EMILIA MATALLANA, Jo!& L. BODRfGUEZ, AND LUIS FRANCO Department of Biochemistry

and Molecular Biology, University of Val&ncia, 46100 Burjassot, Vakncia, Spain Received June 6, 1989; revised September 23, 1989

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jim&z and J. Davies (1980) Nature (London) 287,869-87 I] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-wrn circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRS. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRS. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-1731 for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRS is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nuclcosomes on the promoter can be predicted. especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure. 0 1989 Academic PRESS,h.

The occurrence and significance of nucleosome positioning, i.e., precise location of nucleosomes with respect to DNA sequence, have received much attention in the past few years (see the reviews of Eissenberg et al., 1985; Simpson, 1986). Two main mechanisms to explain nucleosome positioning have been proposed: the existence of regions devoid of nucleosomes that would act as boundaries forcing the adjacent nucleosomes to occupy fixed positions, and the influence of the sequence itself. These mechanisms are not mutually exclusive, and some cases in which both causes seem to be decisive have been described (Bloom and Carbon, 1982; Thoma, 1986). Folding of chromatin may also play a decisive role in nucleosome positioning (Thoma and Zatchej, 1988). Sequence may determine nucleosome positioning either by being recognized by putative nonhistone proteins that could mold the deposition of a histone octamer or by facilitating the wrapping of DNA around the octamer. While no proof of the existence of these sequence-specific nonhistone proteins has 143

been found, there is accumulating evidence that sequence may modulate the mechanical properties of DNA (Calladine and Drew, 1986) that facilitate its bending around a histone octamer (Travers, 1987). Algorithms to localize the sequence-dependent DNA regions that can preferentially interact with histone octamers have been derived (Drew and Calladine, 1987) and they have been favorably tested with experimental data obtained by reconstituting histone octamers onto eukaryotic DNA in vitro (Drew and Calladine, 1987; Kefalas et al., 1988). We have recently demonstrated that nucleosomes can be assembled in vivo on prokaryotic DNA cloned into yeast cells (Estruch et al., 1989). It would be interesting to know whether sequence can determine nucleosome positioning in these instances. To address this issue, we describe in this paper the chromatin structure of transposon Tn903, present in plasmid pAJ50 (Jiminez and Davies, 1980), which can be transformed into yeast cells. This transposon offers the following advantages: (i) its sequence is known (Oka et al., 1981); (ii) 0147-619X/89

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10 mM Tris-HCl, pH 7.4, and they were directly digested without further preparation with either DNase I (Worthington Biochemicals) or micrococcal nuclease (Boehringer Mannheim) as described previously (PerezOrtin et al., 1986a,b). Mapping of the nuclease cutting sites. The nucleasecutting sitesin pAJ50 chromatin were located by the indirect end labeling technique (Wu, 1980). Restriction endonucleases (Boehringer) were used according to manuMATERIALS AND METHODS facturer’s instructions. Electrophoresis, blotYeast strains and transformation. Saccha- ting, hybridization, autoradiography, and romyces cerevisiae strain 689 (Mata, leu2- scanning of the autoradiograms were carried 3,112, ura3-50, canl,lOl) (Zakian, 1981) was out as described elsewhere(Perez-O&i et al., transformed with plasmid pAJ50 following the 1986a). procedure of Hinnen et al. (1978). TransforA map of transposon Tn903, contained in mants were selected for their leu+ phenotype plasmid pAJ50, including the probes used in and they were further screened for resistance this study and the relevant restriction sites is to the antibiotic G418 at a concentration of given in Fig. 1 to facilitate interpretation of 0.2-l .O mg/ml. The APH gene codes for the results. aminoglycoside 3’-phosphotransferase,which phosphorylates the antibiotic G4 18. UntransRESULTS formed cells did not grow even at 0.2 mg/ml G418. Presence of nucleosomes on Tn903 DNA. The number of copies of plasmid pAJ50 in To determine whether the APH gene was ortransformed cells was estimated by a dot-blot ganized into nucleosomes, DNA from microassay (Perez-Ortin et al., 1987); at least 20 coccal nuclease-digested chromatin from copies per cell were found. Restriction of total transformed cells was electrophoresed,blotted, DNA from transformed cells, followed by and hybridized with labeled XS probe (seeFig. electrophoresis, gave a single band hybridiz- 1). The results are given in Fig. 2A, which able with a labeled pBR322 probe, whose mo- clearly shows that nucleosomes actually exist bility correspondedto that of linearized pAJ50 on Tn903 sequences.Five bands with a unit (results not shown). Thesefacts are compatible repeat length ( 165- 170 bp) comparable to that with the idea that the plasmid is not integrated of yeast bulk chromatin can be detected. The in the genome. nucleosomal ladder, however, is somewhatdifGrowth and spheroplasting of yeast. Strain fuse. This is not an artifact due to a poor elec689 transformed with pAJ50 was grown in trophoretical resolution, becausewhen probe YEPG medium (1% yeast extract, 2% bacto- XS was washed out and the same filter was peptone, 2% glucose) containing 0.2 mg/ml hybridized with probe KpnI-EcoRI (KE) from G418. Growth under these conditions indi- the transcribed region of the LEU2 gene, also catesthat the APH gene is being actively tran- present in plasmid pAJ50, a much clearer patscribed. tern was obtained (Fig. 2B) and at least nine Spheroplast formation was carried out as bands could be detected. An identical pattern described by Lohr and Ide (1979). was observed with the chromosomal copy of Nucleasedigestions. Spheroplastswere lysed the LEU2 gene (Martinez-Garcia et al., 1989). in 10 mM NaCl, 3 mM MgC12, 1 mM CaCl*, Although the repeat length of the LEU2 nucleosomes is similar to that found in Tn903, ’ Abbreviations used: IR, inverted repeats in the two the nucleosomal bands in the LEU2 region are much more distinct. It can be argued that borders of Tn903; bp, base pair(s).

it contains the APH gene for kanamycin resistance, which can be expressedin yeast (JimCnez and Davies, 1980); and (iii) the APH gene is flanked by two untranscribed inverted repeats (IRS),’ 1057 bp long. Therefore, the chromatin structure of two untranscribed regions with the same sequence and opposite orientation can be compared with that of the central, transcribed gene.

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-d FIG. 1. Map of transposon Tn903. Two IRS, 1057bp long, representedas open boxes, occur at both ends of the transposon, flanking the central region, which contains the aminoglycoside 3’-phosphotransferase (APH) gene. The restriction sites relevant to this study are indicated: S, SmaI; X, XhoI; H, HindIII. As shown in the upper part of the figure, the positions in Tn903 have been numbered in the direction of the APH gene. The right IR has been also numbered in the opposite direction (italicized numerals) to show the equivalence between the positions in both IRS. In the lower part of the figure, the probes used in this study are identified by letters referring to their restriction ends. The arrows labeled a, b, c, and d indicate the four procedures used for indirect end labeling (seethe text). Sequencedata were taken from Oka et al. (1981).

probe KE would hybridize with DNA from chromosomal LEU2 chromatin but, taking into account that plasmid pAJ50 is present in more than 20 copies (seeMaterials and Methods), it is clear that the pattern shown in Fig. 2B arisesmainly from pAJ50 chromatin. This rules out the possibility that damageto pAJ50 during experimental manipulation is the cause of the diffuse pattern obtained in the Tn903 region (Fig. 2A). Moreover, it could reflect a structural property of Tn903 chromatin, probably the heterogeneity of nucleosomal sizes. Mapping of micrococcal nuclease-sensitive sites in Tn903 chromatin. To determine whether Tn903 nucleosomesare positioned on fixed sequences,we mapped the micrococcal nuclease cutting sites by the indirect end labeling technique (Wu, 1980). Four mapping procedures were used (Fig. 1). Procedure a (restriction with Hind111and probing with SH) was used to map the micrococcal nucleasesensitive sites in the left part of the APH gene, its 5’ flank and the IR. The location of micro-

coccal nucleasecutting sitesbetween the SmaI sites at positions 644 and 1465 was confirmed by procedure b (SmaI restriction and XS probing). The mapping in the right part of the gene, its 3’ flank and the right IR, was accomplished by either procedure c (restriction with XhoI and probing with XS) or procedure d (restriction with SmaI and probing with SH). An example of the results obtained with procedure a is given in Fig. 3. The naked DNA band that maps at about 680, indicated by an arrowhead in lanes a and d of Fig. 3A, is absent in chromatin. The densitogramsshown in Fig. 3B make this difference still clearer. One explanation may be that position 680 is protected in chromatin by the presence of a positioned nucleosome. The spacing between chromatin sites 3 and 4 is 175 bp, compatible with nucleosomal size. The DNA bands at positions 870 and 1330 indicated by dots in Fig. 3, and especially the former, also seem to be somewhat protected in chromatin. The spacingbetween sites4 and 6 is 180 bp and a nucleosome has been ten-

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242-+ 160-+ FIG. 2. Presenceof nucleosomes in Tn903. (A) Autoradiogram of a Southern blot of DNA from micrococcal nuclease-digestedchromatin from yeast cells transformed with plasmid pAJ50, hybridized with labeled XS probe. The seven samples run were digested with increasing amounts of micrococcal nuclease(from right to left). (B) Automdiogram obtained after washingthe filter to remove the XS probe and rehybridizing with a probe from the yeastLEU2 gene, also present in plasmid pAJ50. The lane shown is the left one of (A). The migration of size markers (bp) is given on the left.

tatively centered at position 855 in Fig. 3. Mapping procedure b gave similar results (not shown). Figure 4 gives the results of mapping procedure c. Some noteworthy differences between the patterns of chromatin and naked DNA can be seen. For instance, the naked DNA bands indicated by arrowheads (one in Fig. 4A and three in Fig. 4B) are not present in chromatin and therefore correspond to protected DNA regions. These bands lie between sites 28 and 29,29 and 30, and 30 and 3 1, which are separatedby 175, 155, and 170 bp, respectively. These facts suggestthat three nucleosomes are positioned with their centers

at about 2230, 2400, and 2570. The first two lie on positions symmetrical, within 15 bp, to those centered at positions 680 and 855 detected by procedure a (Fig. 3). Finally, some other minor conclusions are suggestedby Fig. 4. For instance, the DNA band indicated by a dot between sites 21 and 25 is probably not present in chromatin, whereas site 18 seems to be very accessiblein chromatin, asit appears under mild digestion conditions. The spacing between sites 18 and 21 and 21 and 25 is 180 and 160 bp, respectively, compatible with nucleosomal size. At any rate, given the present evidence, we cannot be sure that nucleosomes are positioned on these locations. If they are, their positioning must be more flexible than that of the nucleosomes placed on the IRS. Mapping following procedure d (results not shown) confirms this conclusion. The models depicted in the middle of Figs. 3 and 4 summarize the conclusions on nucleosome positioning. DNase I sensitivity in Tn903 chromatin. Well-defined DNase I-hypersensitive siteswere not found by any of the four mapping procedures. The digestion patterns for both IRS are coincident (results not shown), asthey were in the caseof micrococcal nuclease digestion. This means that the chromatin organization in these regions is sequence-directed. No distinctive features were found in the DNase I digestion of the APH gene,except for the presence of an extended zone, coincident with the promoter of the gene, which is accessible to the enzyme in both naked DNA and chromatin. DISCUSSION

The results given in Fig. 2 show that nucleosomes actually are assembled in vivo on Tn903 sequences.We have previously found that nucleosomes are assembledin vivo on the pBR322 DNA contained in piasmid pAJ50, but, although nucleosomesare not located entirely at random, a true positioning does not exist (Estruch et al., 1989). To determine whether this is a general feature of prokaryotic DNA, we have studied the nucleosome positioning on Tn903. Results

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FIG. 3. Nucleosome positioning in the left IR and the 5’ half of the APH gene following procedure a. (A) Autoradiogram. (B) Densitometric scan of three lanes from (A) between positions 500 and 1500. Lanes a and d give the patterns obtained with naked DNA. The remaining lanes show the patterns of DNA from. chromatin digested with micrococcal nucleaseto varying extents. The mobility of size markers is indicated in the left margin of (A). The arrowheads point to a DNA band that is absent in chromatin samples, and the dots indicate other DNA bandslessclearly protected in chromatin. Bandsin chromatin lanesare numbered consecutively, and those characteristic of chromatin or relevant to explanations of nucleosome positioning are projected onto the map of the studied region, shown in the middle, that outlines the two positioned nucleosomes.The dotted circle representsthe nucleosomefor which the experimental evidence for positioning is weaker.

given in Figs. 3 and 4 allow us to conclude that three positioned nucleosomesexist on the IRS.The distal nucleosomerelative to the APH gene cannot be seen at the resolution level of Fig. 3, but, considering the symmetry of the two detectednucleosomeswith respectto those centered at 2400 and 2230, we can infer that a third nucleosome at about 525, i.e., symmetrical to that of 2570 on the right IR (Fig. 4), would exist on the left. The symmetrical organization of chromatin on both IRS was also detected in the DNase I digestions. It is thus conceivable that the chromatin structure of the IRS depends mostly on the sequence itself. To check this possibility we used the algorithms derived by Drew and Calladine (1987). These algorithms are based

on two lines of evidence. First, by analyzing the sequenceof 177 different DNA molecules from chicken erythrocyte nucleosome cores, Satchwell et al. (1986) found a periodicity in the distribution frequency of di- and trinucleotides that may be correlated with their preferenceto be located in the minor groove when it points out or when it facesthe histone octamer. With these data, matrices of preferred rotational setting were constructed (Drew and Calladine, 1987). A good fit between a given sequence and the matrix indicates that the probability of a nucleosome dyad being centered over this sequenceis high. On the other hand, taking into account that certain oligonucleotide tracts are preferentially found at the ends of the nucleosome cores(Satchwell et al.,

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FIG. 4. Nucleosome positioning in the right IR and the 3’ half of the APH gene. The figure gives the results obtained by procedure c. Electrophoreseswere carried out in 1.8%(A) or 1.O%(B) agarosegels. (A) Lanes a and b, naked DNA (two digestions); c, size markers (1693, 1125, 622, 527,404, 309, 242, and 160 bp); d and e, chromatin (two digestions). (B) Lane a, chromatin; b, naked DNA. The mobility of size markers (B) is indicated by arrows. The dot in lane b (A) indicates a naked DNA band probably absent or very weak in chromatin samples. All the remaining symbols are as in Fig. 3.

1986),a matrix of translational preferencealso can be evaluated (Drew and Calladine, 1987). Figure 5 shows the calculations for the rotational and translational fits between histone octamers and the Tn903 DNA. Only the first 2040 bp have been used in the calculation, as the remaining sequence corresponds to the right IR. From the rotational fit, it can be observed that the deepestminimum (+3.59) occurs at position 694 (or its symmetrical equivalent 2400). The correspondence with the experimental position of the central nucleosome detected in both IRS (680 and 2400) cannot be casual, and we think that this nucleosome is positioned becauseof the extremely favorable bending properties of DNA in this location. Once this nucleosome is fixed, it would

be reasonable to suppose that it determines the positioning of the adjacent nucleosomes. Moreover, translational fit gives two deep minima at both sides, centered at positions 480 and 834 (or their symmetrical equivalents 2605 and 2260), in close agreement (-t30 bp) with the experimental positions found for the two outer nucleosomes on the IRS. Therefore, we think that both rotational and translational sequence signals are acting to determine the location of the three positioned nucleosomes on the IRS. This conclusion is also substantiated by the fact that the values of the minima in Tn903 fits compare very favorably with those obtained with the frog 5 S RNA gene (Drew and Calladine, 1987) and chicken ,L3globin gene (Kefalas et al., 1988).

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FIG. 5. (A) Calculations for rotational (top) and translational (bottom) fits between histone octamers and Tn903 DNA. Data were computed as described by Drew and Calladine (1987). The ordinate scalesare in units of likelihood times RT and range from +2 (rotational) or -1 (translational) for a good fit to -2 (rotational) or -4 (translational) for a bad fit. Positions according to map of Fig. 1 are given on the abscissae. (B) Model for nucleosomepositioning on Tn903 DNA (seethe text). The sequenceis aligned with the figures given in (A). The positioned nucleosomesare depicted as circles.

Figure 5 also shows that a deep (-0.65), broad minimum, centered at x 1100, occurs in the translational fit. Nevertheless, experimental evidence does not sustain the existence of a positioned nucleosome in this region. Moreover, intense bands 8 and 9 (Fig. 3) and the overall sensitivity of this region to DNase I are more compatible with the absenceof nucleosomesaround position 1100. This region coincides with the promoter of the APH gene, which, as previously mentioned, is being expressed under our growth conditions. Larch et al. ( 1987) have shown that RNA polymerase II cannot initiate transcription at a nucleosome, and in two yeast genes, namely, the SUC2 gene (Perez-O&r et al., 1987) and especially the PH05 gene (Almer et al., 1986), it has been shown that positioned nucleosomes that exist in the promoter are removed when the gene is activated. Therefore, it is clear that sequencesignalsdo not suffice for determining nucleosome positioning on active regions, although they can in inactive chromatin, asthat of the IRS.

The nucleosomes on the APH gene do not seemto be positioned, at least not in so strict a manner as they are on the IRS. Sequence determinants are also not too stringent. Several minima with similar values around +2 are seen in the rotational fit for the transcribed region, but they are lessthan one nucleosome length apart (Fig. 5). Moreover, except for a minimum at 1586, the curve for translational fit goes above -1. Thus, many different nucleosome arrangements are equally probable in terms of sequencepreference. The present results show that nucleosome positioning can exist on prokaryotic DNA assembled into chromatin in vivo. Sequencedeterminants seem to be decisive in the caseof the inactive IR regions, but some still-unknown causesuperimposesthesedeterminants on the active promoters. We are engaged in the study of several promoters to shed some light on this problem. ACKNOWLEDGMENTS This work hasbeen supported by Grant PB85-233 from CICYT (Spain). We thank Dr. A. Jimenez for the gift of

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plasmid pAJ50 and Dr. S. Ferrer for the gift of .S.cerevisiue strain 689.

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BLOOM,K. S., AND CARBON,J. (1982). Yeast centromere DNA is in an unique and highly ordered structure in chromosomesand small circular minicromosomes. Cell 29,305-317.

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