topological analysis of plasmid chromatin from yeast and mammalian cells

doi:10.1016/j.jmb.2006.07.015 J. Mol. Biol. (2006) 361, 813–822

COMMUNICATION

Topological Analysis of Plasmid Chromatin from Yeastand Mammalian Cells

Wilbur Tong1, Olga I. Kulaeva1, David J. Clark2 and Leonard C. Lutter1⁎
1Molecular Biology ResearchProgram, Henry Ford Hospital,Floor 5D, One Ford Place,Detroit, MI 48202-3450, USA2Laboratory of MolecularGrowth Regulation,National Institute of ChildHealth and HumanDevelopment, Building 6 Room2A14, National Institutes ofHealth, 6 Center Drive,Bethesda,MD20892-2426,USA
E-mail address of the [email protected]

0022-2836/$ - see front matter © 2006 E

Yeast has proven to be a powerful system for investigation of chromatinstructure. However, the extent to which yeast chromatin can serve as amodel for mammalian chromatin is limited by the significant number ofdifferences that have been reported. To further investigate the structuralrelationship between the two chromatins, we have performed a DNAtopological analysis of pRSSVO, a 5889 base-pair plasmid that can replicatein either yeast or mammalian cells. When grown in mammalian cells,pRSSVO contains an average of 33 negative supercoils, consistent with onenucleosome per 181 bp. This is close to the measured nucleosome repeatlength of 190 bp. However, when grown in yeast cells, pRSSVO contains anaverage of only 23 negative supercoils, which is indicative of only onenucleosome per 256 bp. This is dramatically different from the measurednucleosome repeat length of 165 bp. To account for these observations, wesuggest that yeast chromatin is composed of relatively short ordered arraysof nucleosomes with a repeat of 165 bp, separated by substantial gaps,possibly corresponding to regulatory regions.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: chromatin; DNA topology; nucleosome; nucleosome repeatlength; supercoiling
*Corresponding author
The DNA of eukaryotes is organized into chroma-tin, of which the basic repeating subunit is thenucleosome. The nucleosome core is a compactstructure at the center of which is the histone octamercomposed of two copies each of histones H2A, H2B,H3, and H4. Wrapped onto the octamer surface is146 bp of DNA in the form of 1.75 left-handedsuperhelices. This wrapping, combined with changesin the screw of the DNA upon binding to thenucleosome, induces a change in the topologicallinking number of circular DNA when nucleosomesare assembled in the presence of a topoisomerase.1,2

The magnitude of this change measured for theminichromosomes of simian virus 40 (SV40) inmammalian cells is –1/nucleosome,3–7 and this issupported by studies of nucleosomes reconstituted invitro.8 Measurement of this chromatin-induced link-ing number change is therefore a convenient meansfor assessing the density of nucleosomes assembledonto circular DNA in a cell, and thus is useful inanalysis of chromatin assembly and structure.

ng author:

lsevier Ltd. All rights reserve

A second indicator of nucleosome density is nu-cleosome repeat length, which is measured byanalysis of a nuclease digest of chromatin.9 For ex-ample, micrococcal nuclease cleaves preferentiallybetween nucleosome cores in chromatin, so analysisof the sizes of DNA fragments in a partial digest re-veals the spacing between nucleosomes in chromatin.This is termed the nucleosome repeat length, whichimplies a specific nucleosome density. However, it isimportant to realize that the topology and nucleasedigestion methods measure distinct but complemen-tary properties of chromatin: the topology reflects theoverall nucleosome density, but cannot detect possi-ble local heterogeneity of densities contributing tothat global value. The micrococcal digest measuresthe density of a section of chromatin, but only if nu-cleosomes are in regular arrays of a constant spacing.Even in this case, the measurement is necessarily ameasurement of a subfraction of the total, so it doesnot quantify the overall nucleosomedensity. If there islocal heterogeneity in densities, then the assumptionthat the micrococcal result is representative of theglobal nucleosome density will be incorrect.A large number of studies using micrococcal

nuclease have revealed that the nucleosome repeat

d.

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814 Yeast and Mammalian Chromatin Structures

length is different for various organisms and tissues.For example, for mammalian tissue-culture cells thelength is 190 bp/nucleosome, while for yeast thevalue is 165 bp/nucleosome.9,10 This indicates thatnucleosomes are packed more densely overall inyeast than in mammalian cells. However, noindependent assessment has been performed tocorroborate this difference in such a fundamentalproperty of the two chromatins.We address this question here by measuring the

chromatin-induced linking number change of a DNAcircle replicated in yeast cells and comparing it to thatof the sameDNAcircle replicated inmammalian cells.The topological results indicate that the nucleosomedensities of yeast and mammalian chromatin doindeed differ, but in the sense opposite to thatpredicted by the nucleosome repeat length.

Measurement of plasmid supercoildensities in mammalian cells

An initial study was performed to investigate therange of variation of the value for the chromatin-duced supercoil density in mammalian cells. Thesupercoil density of the SV40 minichromosome(–0.0523, see Table 1) has been measured directly

Table 1. Summary of topological results

Cell type Plasmida Size (bp) ΔL

COS: pRSSVO 5889 −32.6pTEKO 3512 −19.5pOS47 3175 −17.8pOS67 1568 −8.3

BS-C-1: SV40f 5243 −26.1Yeast: pRSSVOg 5889 −23

pRSSVOh 5889 −23.6TACi 2468 −12

TA-HIS3j 2435 −10.5TRP1ARS1k 1453 −7

a Construction of plasmids. Plasmid pRSSVO was constructed asreplication (nucleotides 5161 to 74) was obtained by PCR using pOS3

5′-GCGCGCGGATCCCATCTTTGCAAAGCTTTTTGC

5′-GCGCGCGGATCCCTAACTCCGCCCAGTTC

(BamHI sites underlined and SV40 sequences in bold). This fragmentpolylinker of pRS426 (Stratagene), which is a high-copy yeast vector cURA3 (1085 bp yeast sequence) as selection marker to obtain pRSSVOin the SV40 origin was closest to the EcoRI site in the pRS426 polylinkgenerated by adding ApaI and BstXI sites to the sequence spanning nuccloned into pGEM-T Vector (Promega). Plasmid pOS37 was derived fPlasmid pOS47 was generated by adding BstXI and ApaI sites to thewhich the fragment was cloned into pGEM-T Vector. Plasmid pOS67spanning nucleotides 5163 to 76 of SV40 by PCR, after which the fragmBstUI site. Plasmid pSVTCMV (kindly provided by Dr Michael Impedownstream of the CMV promoter of pBK-CMV (Stratagene).

b Supercoil density=(ΔL/plasmid size)×(10.5 bp/turn). The expecc The nucleosome repeat length predicted from the topological mead Measured from a micrococcal nuclease digest of host cell chromae The ratio of the nucleosome repeat length predicted from topologf From Drabik et al.5g Grown in selective medium.h Grown in YPD medium.i From Shen et al.42j From Kim & Clark.40k From Thoma et al.33 and from Pederson et al.46

using the topoisomer band counting procedure,the most rigorous method available. There hasbeen a report of a supercoil density of –0.038 for atransfected plasmid,11 but we have found theindirect measurement method used in that studycan be as much as 50% in error when compared tothe standard direct measurement method usedhere, i.e. with the relaxed and chromatin-derivedforms related by a connector series. However, ithas been reported that supercoil density can varywith SV40 subpopulations.12,13

To clarify this issue, we performed direct mea-surements for several different plasmids transfectedinto mammalian (COS) cells in order to evaluatepossible variability in the values for chromatin-induced linking number change and supercoildensity. SV40 origin sequences were cloned intofour plasmids of varying sizes and sequences (seeTable 1). The SV40 origin allows the plasmid to bereplicated in mammalian cells when SV40 T-antigenis present, as for example in COS cells.14 It isimportant that the plasmid be replicated, so thatchromatin is assembled onto it via the normalreplication-dependent assembly process of thecell.15,16 The plasmids were transfected individuallyinto COS cells using a new procedure that generates

σb bp/(−ΔL)c Repeatd Ratioe

−0.0581 181 190 1.05−0.0583 180 190 1.06−0.0589 178 190 1.07−0.0556 189 190 1.01−0.0523 201 190 0.95−0.041 256 165 0.64−0.0421 251 165 0.66−0.0511 205 165 0.8−0.0453 231 165 0.71−0.0506 208 165 0.79

follows. A 181 -bp DNA fragment containing the SV40 origin of7 (see below) as template and the following primers:

was digested with BamHI and inserted into the BamHI site in theontaining a 2 μ origin of replication (1347 bp yeast sequence) and(5889 bp).44 The 163 bp insert was oriented such that nucleotide 74er. The sequence of the insert was verified. Plasmid pTEKO wasleotides 5032 to 299 of SV40 by PCR, after which the fragment wasrom plasmid pSV11a by deletion of the EcoRV/SwaI fragment.45

sequence spanning nucleotides 5163 to 76 of SV40 by PCR, afterwas generated by adding EcoRV and XbaI sites to the sequenceent was blunt cloned into plasmid pJRtac99 (ATCC #87018) at theriale, University of Michigan) contains SV40 T-antigen sequences

ted result for yeast is σ=–1/(165/10.5)=–0.064.surement.tin.9

y divided by that measured from a micrococcal nuclease digest.

Figure 1. Topological analysis of plasmids from COScells. (a) Two-dimensional electrophoresis of pOS67 fromCOS nuclear extract. Plasmid pOS67 was purified follow-ing transfection into COS cells and analyzed by two-dimensional electrophoresis in Tris–borate/EDTA (TBE)buffer in a manner similar to that described.5,37,38 Fol-lowing electrophoresis, the gel was stained with ethidiumbromide and imaged (FMBIO). “Connector” samples wereprepared by incubating pOS67 with topoisomerase II anda series of concentrations of chloroquine diphosphateranging from 0 to 700 μg/ml at 37 °C.38 The firstdimension (TBE plus 0.35 μg/ml of chloroquine dipho-sphate) is from top to bottom, while the second dimension(TBE plus 2.7 μg/ml of chloroquine diphosphate) is from

815Yeast and Mammalian Chromatin Structures

high levels of replicated plasmid minichromosomes(see the legend to Figure 1(a)). Nuclear extract wasprepared and the DNA analyzed by two-dimen-sional electrophoresis to measure the chromatin-induced linking number change.5

An example is shown in Figure 1(a) for theplasmid pOS67. At the left is the topoisomerdistribution of the plasmid as isolated from nuclearextract, i.e. when it was in its chromatin form. At theright is the distribution of the plasmid followingrelaxation as purified DNA in an isotonic buffer. Inthe center is the distribution of a mixture of“connector” samples relaxed as bare DNA in thepresence of various amounts of chloroquine dipho-sphate. Such a connector series is essential for anybut the smallest circles in order to quantify the

left to right. The loading order of the samples was, fromleft to right: nuclear extract, connector, and bare relaxedDNA. The linking number increases in a clockwisedirection around the arc of the topoisomers. One topo-isomer has been arbitrarily assigned a value of 0 and theother topoisomers have been numbered accordingly. Thenicked species of each of the respective samples areindicated by N. At the lower left is a densitometer scan ofthe nuclear extract sample, while at the lower right is ascan of the bare relaxed DNA sample. Plasmid transfec-tion and sample preparation. Plasmid pOS67 was trans-fected into COS-7 cells as follows. COS-7 cells15 wereseeded at 50% confluency 4 h before transfection. Fortransfection of one 15 cm plate of cells, a mixture of 40 μl ofLipofectamine (GIBCO) and 1.56 ml Optimem (GIBCO)was incubated for 20 min at room temperature. Themixture was then combined with 1.6 ml of Optimemcontaining 0.5 μg of pOS67 and 0.5 μg of pSVTCMV, andincubated for 30 min at room temperature. Cells werewashed once with Optimem, after which 12.8 ml ofOptimem and 3.2 ml of the Lipofectamine-DNA-Optimemmixture were added to the plate. Cells were incubated for18 h, after which the Optimem was replaced withstandard medium. Nuclear extract was prepared at 72 hpost infection.18 The extract was incubated at 37 °C for30 min, after which the DNA was extracted with phenolbefore electrophoresis. Plasmid pOS67 and other plasmidswith comparable origin sequences (see Table 1) replicateto levels of about 0.2 μg/plate when additional T-antigen is supplied by pSVTCMV. At this level, thepRSSVO DNA can be readily detected by ethidiumstaining. (b) A map of pRSSVO. (c) Two-dimensionalelectrophoresis of pRSSVO from COS nuclear extract.Plasmid pRSSVO was purified following transfection intoCOS cells and analyzed by two dimensional electrophor-esis along with samples of connector DNA that wererelaxed in the presence of varying amounts chloroquine(see (a)). The first dimension (TBE plus 0.65 μg/ml ofchloroquine diphosphate) is from top to bottom, while thesecond dimension (TBE plus 2.9 μg/ml of chloroquinediphosphate) is from left to right. An image of theethidium-stained gel is shown. The topoisomer distribu-tions of the following samples are indicated: NE, nuclearextract; R, a sample relaxed with no chloroquine; 700,samples relaxed with 700 μg/ml of chloroquine. The tworemaining distributions are those of connector samples,with the linking number increasing in a clockwisedirection around the arc formed by the topoisomers.One topoisomer has been arbitrarily assigned a value of 0and the other topoisomers have been numbered accord-ingly. The nicked species of the different samples areindicated by N.

Figure 2. Topological analysis of pRSSVO from yeastcells. (a) Two-dimensional electrophoresis of pRSSVOgrown in yeast and COS cells. Yeast strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (ATCC #4001298)39 wastransformed with pRSSVO; transformants were selectedon plates lacking uracil. A transformant was grown tohigh density in non-selective medium (YPD) and DNAwas purified by direct extraction of spheroplasts with SDS

40


topological linking number difference between thebare relaxed and chromatin-bound forms of theDNA.This difference is termed the chromatin-inducedlinking number change. The densitometer scansbelow the gel image show that the center of the bareDNA distribution is at –1.8, while that of thechromatin-bound distribution is –10.1. Thus, thechromatin-induced linking number change ΔL iscalculated as:

DL ¼ Lchromatin � Lbare relaxed ¼ �10:1� ð�1:8Þ¼ �8:3

This corresponds to a supercoil density of –0.0556(see Table 1), a value that quantifies the specific levelof supercoiling.The same analysis was performed for plasmids

pTEKO, pOS47, and pRSSVO. A map of pRSSVO isshown in Figure 1(b). It contains the SV40 origin of

replication inserted into the plasmid pRS426, whichis a pUC19-derived plasmid containing a yeast 2 μplasmid origin of replication that allows its replica-tion in yeast cells with a high copy number (seebelow). It also contains the yeast URA3 gene as aselection marker, with the result that 41% of theplasmid is yeast sequence.The topological analysis of pRSSVO grown in

COS cells is shown in Figure 1(c). The topoisomerdistribution of the purified plasmid relaxed in vitro is

as described. Samples of plasmid pRSSVO replicated inyeast or COS cells were analyzed by two-dimensionalelectrophoresis.40,41 The first dimension (Tris–phosphate/EDTA buffer (TPE) plus 10 μg/ml of chloroquine dipho-sphate) is from top to bottom, while the second dimension(TPE plus 20 μg/ml of chloroquine diphosphate) is fromleft to right. Note that the concentrations of chloroquinediphosphate needed for the gel run with TPE here aremuch higher than those needed for the gel run in TBE (seeFigure 1(a), (c)). This is because the affinity constant for thebinding of chloroquine to DNA differs in the two types ofbuffer. Following electrophoresis, the gel was blotted toGeneScreen Plus membrane (Perkin-Elmer). To avoiddetecting the endogenous yeast 2 μ plasmid, the probeused was generated from pNEB-URA3 (which contains no2 μ sequences) by random priming. Plasmid pNEB-URA3(3832 bp) contains the URA3 gene inserted into a pUCbackbone,42 so it will hybridize with about 65% of thepRSSVO sequence but not with sequences of the endo-genous yeast 2 μ plasmid. An autoradiograph of theprobed blot is shown. The samples were DNA purifiedfrom: Y, whole cell extract of transfected yeast cells grownin YPD; C, nuclear extract from transfected COS-7 cells, asin Figure1(c), NE, the linking number increases in aclockwise direction around the arc formed by thetopoisomers. The nicked species of the various samplesare indicated by N. (b) One-dimensional analysis. PlasmidpRSSVO was purified from yeast cells as described for (a)above, after which samples were fractionated by electro-phoresis in a 0.7% (w/v) agarose gel containing 5 μg ofchloroquine diphosphate/ml using TPE as the runningbuffer with recirculation; 42 V, 6 h, as described.43 The gelwas blotted and probed as in (a). Lanes A and B containDNAs from two separate transformants that were grownin selective (synthetic complete medium lacking uracil)medium. Lanes C and D contain DNAs from two separatetransformants that were grown in non-selective (YPD)medium. The sample in lane E is the same as that marked700 in Figure 1(c), and the topoisomer corresponding to–28 in the numbering system in Figure 1(c) is indicated atthe right. Topoisomers are negatively supercoiled underthese gel conditions, so linking number increases withdecreasing mobility. Below are shown the densitometricscans for the indicated lanes, with high mobility at theright. F is the scan of the nuclear extract sample markedNE in Figure 1(c). The mobility of the nicked DNA (N) isindicated at the left of the autoradiograph, while thearrowhead designates the center of the topoisomerdistribution in scan C.


indicated by R at the right. Its center is located at–1.0 using the numbering system indicated. Thetopoisomer distribution of the nuclear extract sampleis indicated by NE at the left. The center of thisdistribution is –33.6, as determined from the scan ofthe distribution (see Figure 2(b)F). The linkingnumber change induced by assembly of chromatinonto pRSSVO in mammalian cells is therefore:

DL ¼ �33:6� ð�1:0Þ ¼ �32:6

The corresponding supercoil density is –0.0581.The average of the values for the four plasmids(Table 1) is –0.0577±0.0014. This is somewhat morenegative than the value of –0.0523 measured forSV40,5 but the difference is relatively small. Thus, theSV40 value that has been widely used to representchromatin-induced supercoiling is close to theaverage value, which exhibits a minor variation.

Comparing the supercoil densities ofthe same plasmid grown in yeast andmammalian cells

With measurements of the value and variation ofthe mammalian cell supercoil density in hand,chromatin-induced supercoil densities in yeast andmammalian cells were compared directly by analyz-ing the same plasmid in the two types of cells.Plasmid DNA extracted from yeast cells containingpRSSVO was compared directly with that frompRSSVO-transfected COS cells by two-dimensionalelectrophoresis. The blot of the gel shows that thecenter of the topoisomer distribution of the plasmidgrown in yeast cells (Figure 2(a)Y) is locatedconsiderably further in a clockwise direction aroundthe topoisomer arc than that from COS cells (Figure2(a)C). The linking number increases in a clockwisedirection around the arc in this gel, so this indicatesthat the mean linking number in yeast is substan-tially greater than that in COS cells. Thus, this directcomparison of the same plasmid grown in the twocell types demonstrates that yeast chromatininduces substantially fewer negative supercoilsthan does mammalian chromatin.The amount of difference between the two sam-

ples was then quantified. The breadth and curvatureof the yeast distribution in the two-dimensionalseparation in Figure 2(a)Y made scanning difficult,so a one dimensional separation was used togenerate scans of the yeast distribution for quanti-fication. Figure 2(b) shows the results of growingpRSSVO-containing yeast cells in selective medium(Figure 2(b)A and B)as well as non-selectivemedium (Figure 2(b)C and D). The topoisomerdistributions in the autoradiographs at the top ofFigure 2(b) indicate that there is little difference inthe centers of the topoisomer distributions, and thescans at the bottom of the Figure confirm this.Quantification of the topological difference betweenpRSSVO grown in yeast and mammalian cells wasdetermined from the yeast one-dimensional datathrough the use of a pRSSVO “connector” sample(Figure 2(b)E; Figure 1(c) 700) that was relaxed in the

presence of 700 μg/ml of chloroquine diphosphate.The inclusion of this standard relates the topoisomerdistributions of the yeast samples to those of theCOS sample and the samples relaxed in vitro fromFigure 1(c). Using the assigned numbering register,it can be seen in the scans at the bottom of Figure 2(b)that the center of topoisomer distribution of thebasal transcription yeast sample in Figure 2(b)C is at–24.6, which calculates to a chromatin-inducedlinking number change:

DL ¼ �24:6� ð�1:0Þ ¼ �23:6

This is strikingly lower than the value of −32.6measured above for the same plasmid in COS cells.Accordingly, the corresponding supercoil density of–0.041 is substantially less negative than the valuesfor any of the other plasmids grown in COS cells (seeTable 1). To our knowledge, this is the first topo-logical analysis of the same plasmid replicated inyeast and mammalian cells, and the results indicatethere is a significant difference in chromatin structuresassembled on the plasmid in the two types of cells: theyeast nucleosome density is substantially lower.As discussed above, the nucleosome repeat

length, measured from amicrococcal nuclease digestof nuclei,9 can be used to calculate the chromatin-induced linking number change for a plasmid repli-cated in a given cell. Thus micrococcal nucleasedigestion of COS cell nuclei indicates that there are190 bp of DNA per nucleosome, so pRSSVO is cal-culated to contain 5889 bp/(190 bp/nucleosome)=31 nucleosomes. A study of chromatin assembled invivo has measured a value of ΔL=−1.0/nucleo-some,5 and this value is supported by an in vitrostudy of reconstituted nucleosomes.8 Thus, pRSSVOgrown in COS cells is predicted to have a chromatin-induced linking number change of ΔL=–31. Thevalue of –32.6 measured here is consistent with thiscalculation (see Table 1).However, the same calculation done for the yeast

result reveals a substantial discrepancy. Micrococcaldigestion indicates that yeast contain 165 bp/nucleo-some, which predicts a value of ΔL=–35.6. Thevalues of –23.6 and –23.0 measured here (Table 1) areonly two-thirds of that. Thus, the value predictedfrom micrococcal nuclease digestion for the chroma-tin-induced linking number change in mammaliancells corresponds reasonably well with that observed.In marked contrast, the value predicted for yeast isconsiderably higher than that for mammalian cells,but the measured value is substantially less.

Micrococcal nuclease digestion ofpRSSVO in yeast nuclei

A possible explanation for this discrepancy couldbe that plasmid pRSSVO differs from other plasmidsthat have been analyzed in yeast by having a nucleo-some repeat length that is longer than 165 bp. Thelinking number measured would predict a repeatlength of about 250 bp. To test this possibility, nucleifrom yeast containing pRSSVO were digested withmicrococcal nuclease, after which the extracted


DNA was fractionated by agarose gel electrophor-esis. Figure 3 (lower left) shows the ethidium-stained gel, representing the bulk yeast chromatin,as well as an autoradiograph of the blot of the gelprobed with DNA from pRSSVO (upper left panel),representing pRSSVO chromatin. The bands repre-senting micrococcal cleavage between nucleosomesare indicated by arrowheads. It can be seen in bothpanels that the size of the third band is just under500 bp, while that of the sixth band is just under1000 bp. These sizes correspond to a nucleosome

Figure 3. Micrococcal nuclease digestion of yeast nuclei.micrococcal nuclease, after which the extracted DNA was sepblotted and probed. The upper left panel shows an autoradiogrbeneath is an image of the ethidium-stained gel. Digests includnuclease: A, 0; B, 2; C, 4; D, 8; and E, 16. A radioactively labeamount of the same marker DNA is in lane G. Fragment siznucleosome ladder are indicated by arrowheads. The panels ingel on which were separated two sets of samples of the sameprobed with pUC19 (center panel) andURA3 (right panel) as inrespective gels of the digestion sample containing the highestblot is to the left of the scan). The upper scan is from the blot ppUC, and the lower is from that probed byURA3. The arrowhemark the sixth band in the micrococcal ladder. Digestion detailfrom 700 ml of BY4741::pRSSVO cells grown in YPD medium5263) was used to prepare the spheroplasts. The nuclei were rMgCl2, 0.5 mM CaCl2, 5 mM 2-mercaptoethanol with proteasand divided into five 400 μl aliquots. Nuclei were warmed to 30was added to 0, 2, 4, 8 or 16 units/ml. Digestion was for 5 miEDTA to 10 mM, SDS to 2% (w/v) and potassium acetate tovolume of chloroform, precipitated with 0.7 vol. isopropanolEDTA with RNase A at 10 μg/ml. Samples were analyzed inblotted to GeneScreen Plus membrane (Perkin-Elmer) and pro

repeat length of approximately 150–160 bp. Thus,the repeat length of a micrococcal nuclease digest inyeast is the same for both the bulk and pRSSVOchromatins. Importantly, the plasmid repeat is notthe 250 bp predicted above for the lower nucleo-some density (Table 1).The relatively high background in the autoradio-

graph in Figure 3 (upper left) suggested that portionsof pRSSVO might differ in the regularity of nucleo-some arrays they contain. To further investigate this,separate blots of the same digest were probed with

Yeast nuclei were digested with increasing amounts ofarated by electrophoresis in an agarose gel. The gel wasaph of a blot probed with pNEB-URA3, while immediatelyed the following concentrations (units/ml) of micrococcalled marker DNA is in lane F, while an ethidium-stainablees (in base-pairs) are indicated at the right. Bands of thethe upper right are autoradiographs of blots of a separatemicrococcal digest series, only in this case the blots weredicated. In the lower right panel are scans of the lane in thelevel (16 units/ml) of micrococcal nuclease (the top of therobed by pNEB-URA3, the middle is from that probed byads at the side of the URA3 blot and above the URA3 scans are as follows. Yeast nuclei were prepared as described42

to an absorbance at 600 nm of 0.9; lytic enzyme (Sigma L-esuspended in 2 ml of 10 mM Hepes-K (pH 7.5), 0.5 mMe inhibitors (Roche) supplemented with 5 nM pepstatin A°C for 2 min and thenmicrococcal nuclease (Worthington)n at 30 °C. Reactions were stopped by the addition of Na-1 M. DNAwas purified by two extractions with an equaland dissolved in 50 μl of 10 mM Tris–HCl (pH 8.0), 1 mMa 1.5% agarose/Tris-acetate/EDTA (TAE) gel, which wasbed as described for Figure 2(a).


portions of pRSSVO derived from Escherichia coli(Figure 3, pUC19) or from yeast (Figure 3, URA3).The results show that the 165 bp ladder is moreclearly defined when the blot is probed with theyeast URA3 sequence (Figure 3, URA3) than when itis probed with E. coli sequence (Figure 3, pUC). Thisis confirmed in scans shown in the lower panel of therightmost lane of each blot. Six bands are clearlyapparent in theURA3-probed blot, while the pUC19-probed blot exhibits a ladder that is not as welldefined due to a much higher background smear.These results indicate that the yeast sequence isbetter organized in arrays of nucleosomes than is theE. coli sequence. It suggests that at least part of thereason for the lower number of nucleosomes on theplasmid grown in yeast cells may be due to aninability of yeast to assemble efficiently nucleosomalarrayswith a 165 bp repeat ontoE. coli sequences. Thisinefficiency could be related to the absence fromE. coliDNA of signals associated with nucleosomebinding.17 Again, it should be noted that the E. colisequences were assembled efficiently into nucleo-somes in COS cells (Table 1).

Relation to other studies

The linking number change of –23 for pRSSVO inyeast is less than that predicted from the 165 bprepeat length, and it is substantially less than thevalue of –32.5 measured for the same plasmid inmammalian cells. Such a difference in the chroma-tin-induced linking number change between twotypes of chromatin can result from a difference in thenumber of nucleosomes, a difference in the linkingnumber change induced per nucleosome, or both.18An explanation for the source of this differencecould lie in the numerous structural differences thathave been reported between yeast and mamma-lian chromatin.19,20 For example, a possible diffe-rence in the linking number change per nucleosomemight be deduced from two reported results: hy-peracetylation reduces the linking number changeper nucleosome reconstituted on DNA in vitro,21 andthere is higher histone acetylation in yeast chroma-tin than mammalian chromatin.22 However, adirect study18 demonstrated that the topologicaleffect observed in vitro21 was found not to occur innative chromatin assembled in vivo, making unli-kely this explanation for the topological differencebetween yeast and mammalian chromatins.Another possible explanation for the difference isthat yeast and mammalian core histones differ insequence. However, while a comparison of thecrystal structures of nucleosomes reconstitutedwith histones with yeast20 or metazoan23 sequencesindicates subtle differences, there are not the clearchanges in wrapping or duplex twist that would beneeded to account for the large topological differ-ence observed here. Yet another difference betweenyeast and mammalian chromatins is the lower levelof histone H1 in yeast: one study finds yeast H1 ispresent at about one copy per 37 nucleosomes,24

while another reports one copy per four nucleo-somes.25 It has been proposed that the absence ofH1 frommammalian transcribing chromatin causesa reduction in the amount of the negative linkingnumber change per nucleosome.26 However, thiswas shown not to be the case by a direct analysis ofnative mammalian chromatin, where removal ofH1 was found to have a negligible effect on thelinking number of SV40 chromatin.18,27 Finally,studies in vitro indicate that the process oftranscription disrupts nucleosomes, which couldlead to a reduction in chromatin-induced linkingnumber change if the disruption persists longenough.28 However, little difference was foundfor pRSSVO under selective or non-selective con-ditions (Table 1). Moreover, a direct comparison ofthe chromatin-induced linking change of transcrib-ing and non-transcribed chromatin shows nodifference in either mammalian5,29 or yeastcells.30 In summary, a survey of the knownstructural differences between yeast and mamma-lian chromatin provides no clear indication that theobserved topological difference is due to a differ-ence in the linking number change per nucleosome.Instead of a difference in the linking number pernucleosome, we propose that the different valuesfor chromatin-induced linking change is due tofewer nucleosomes on pRSSVO from yeast com-pared to that from mammalian cells.

Variation in nucleosome density inchromatin

The topological results and the digestion resultsare in reasonable agreement in their estimates ofnucleosome density in mammalian cells. However,this agreement contrasts markedly with the situa-tion in yeast cells. Thus, the value of –23 obtained forpRSSVO in yeast cells is only two-thirds of the –35.6value predicted from the 165 bp nucleosome repeatlength determined from micrococcal nucleasedigests of yeast chromatin.9,10,31 This observationfor pRSSVO is not atypical of yeast plasmidsbecause quantitatively similar results have beenobtained for several yeast plasmids (Table 1).To explain the discrepancy, we propose the

difference arises from two sources. First, for thediscrepancy observed in the yeast plasmids (TA-HIS3, TAC, TRP1ARS1, which are composedentirely of yeast DNA; Table 1) as well as in theyeast portion of pRSSVO, we propose that nucleo-somes are assembled into short arrays with a 165 bprepeat and gaps corresponding to nucleosome-freeregulatory regions. This argument is based on arecent analysis of the nucleosome density of asignificant proportion of the entire yeast genome,32

which indicates that although most nucleosomes areorganized into arrays with the expected shortspacing, the remainder of the chromatin is appar-ently less ordered. In particular, gene promotersappear to be generally nucleosome-free gaps ofabout 200 bp. This fits with many previous studies


of specific yeast genes suggesting that nucleosomesare disrupted or displaced at the promoter butrelatively well-ordered on the gene itself. In the caseof pRSSVO, the gaps might correspond to the URA3promoter and the 2 μ replication origin. In the casesof the TRP1ARS1 and TA-HIS3 plasmids, such gapshave been mapped.33,34

Second, for the E. coli sequence portion ofpRSSVO, we propose that nucleosomes areassembled inefficiently on E. coli sequences inyeast, resulting in a markedly lower nucleosomedensity on these regions. This would account for thefinding that pRSSVO contains a nucleosome densitymeasured by topology that is lower than even theyeast plasmids (Table 1).The finding here that the overall nucleosome

density is less than the 165 bp per nucleosomeobserved in the bulk digests is supported by otheryeast studies. Electron microscopy of yeast minichro-mosomes indicates considerably fewer nucleosomesthan would be predicted for a 165 bp nucleosomerepeat length (Dr C. Woodcock, personal commu-nication). In addition, there is an indication that theplasmid findings here indicating reduced nucleosomedensity also extend to the yeast chromosome. A2250 bp circle excised from the yeast chromosome byrecombination is reported to contain 12nucleosomes,35 although it was unclear how thenumber of nucleosomes was determined (e.g.whether the nucleosome number was measured bythe topological method such as that used here or bysome other method). That value calculates to onenucleosome per ∼190 bp, which is again significantlyless than the density predicted from the 165 bpnucleosome repeat length. This indicates that thereduced density is not limited to plasmid chromatin,but is characteristic of the chromosome as well.A simple calculation serves to illustrate the

effects of nucleosome-free gaps on interpretationof the repeat length data: Yeast genes are typicallyabout 1 kb in length; a 1 kb open reading framecan accommodate six nucleosomes with a repeatlength of 165 bp. If 200 bp of nucleosome-freepromoter DNA is added to the calculation, therewill be six nucleosomes on 1.2 kb, predicting a200 bp average repeat. Thus, our results can beaccounted for, in principle, by arguing that inyeast there are short nucleosomal arrays with the165 bp repeat on the genes and nucleosome-freegaps at the promoters. This predicts micrococcalnuclease repeat lengths of 165 bp that fade away ataround 6-mer or so, as observed for the URA3gene in pRSSVO. In contrast, the genes of highereukaryotes are generally much longer, predictingfewer nucleosome-free gaps and longer orderedarrays, resulting in longer nucleosomal arrays anda nucleosome count much closer to that expectedfrom their spacing.While the nucleosome repeat length of most types

ofmammalian cells has beenmeasured to be between190 bp and 200 bp,9 there are exceptions. For example,neuronal nuclei isolated from the cerebral cortex ofrabbit have a nucleosome repeat length of 160 bp.36 To

our knowledge, topological studies such as thosereported here have not been performed on theseneuronal cells, although such experiments would beof interest. However, this result raises the possibilitythat even though yeast chromatin differs significantlyfrom the bulk chromatin of most mammalian cells aswell as from that of the transcribing fraction ofmammalian chromatin (see above), it may never-theless be similar to some other subfraction of bulkchromatin or to the chromatin of certain specializedmammalian cell types.In conclusion, a nuclease digestmeasures the inter-

nucleosome distance in stretches of nucleosomes thatare regularly spaced. This distance is different inyeast (165 bp) and mammalian cells (190 bp). Whiledigestion results provide a direct measurement ofthe nucleosome density in those ordered stretches,they do not provide the average nucleosome densityfor a stretch of chromatin that is not composedentirely of such regularly spaced nucleosomes. Thisaverage density is measured by topological analysis.These points are illustrated by the findings here thatthe overall nucleosome density is greater in aplasmid when it is grown in mammalian cellscompared to when it is grown in yeast cells, eventhough the nucleosome repeat length measured inyeast indicates a higher density for the regularlyspaced nucleosomes. Thus yeast chromatin differsfrom mammalian chromatin in the repeat length ofordered arrays, as previously shown, but also in theoverall density of nucleosomes in its chromatin.

Acknowledgements

This work was supported by Public Health Servicegrants GM-56216 and GM-49988 from the NationalInstitute of General Medical Sciences (LCL) and, inpart, by NIH (NICHD) intramural funds.

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Edited by J. O. Thomas

(Received 20 January 2006; received in revised form 23 June 2006; accepted 7 July 2006)Available online 15 July 2006

topological analysis of plasmid chromatin from yeast and mammalian cells

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