Proc. Natl. Acad. Sci. USAVol. 93, pp. 1370-1375, February 1996Biochemistry

Mapping nucleosome position at single base-pair resolution byusing site-directed hydroxyl radicalsANDREW FLAUS, KAROLIN LUGER, SONG TAN, AND TIMOTHY J. RICHMOND*Institut fur Molekularbiologie und Biophysik, Eidgenossiche Technische Hochschule, Honggerberg, CH-8093 Zurich, Switzerland

Communicated by Kurt Withrich, Eidgenossiche Technische Hochschule, Zurich, Switzerland, October 11, 1995

ABSTRACT A base-pair resolution method for determin-ing nucleosome position in vitro has been developed to com-plement existing, less accurate methods. Cysteaminyl EDTAwas tethered to a recombinant histone octamer via a mutanthistone H4 with serine 47 replaced by cysteine. When assem-bled into nucleosome core particles, the DNA could be cut sitespecifically by hydroxyl radical-catalyzed chain scission byusing the Fenton reaction. Strand cleavage occurs mainly at asingle nucleotide close to the dyad axis ofthe core particle, andassignment of this location via the symmetry of the nucleo-some allows base-pair resolution mapping of the histoneoctamer position on the DNA. The positions of the histoneoctamer and H3H4 tetramer were mapped on a 146-bp Lyte-chinus variegatus 5S rRNA sequence and a twofold-symmetricderivative. The weakness of translational determinants ofnucleosome positioning relative to the overall affinity of thehistone proteins for this DNA is clearly demonstrated. Thepredominant location of both histone octamer and H3H4tetramer assembled on the 5S rDNA is off center. Shifting thenucleosome core particle position along DNA within a con-served rotational phase could be induced under physiologi-cally relevant conditions. Since nucleosome shifting has im-portant consequences for chromatin structure and gene reg-ulation, an approach to the thermodynamic characterizationof this movement is proposed. This mapping method ispotentially adaptable for determining nucleosome position inchromatin in vivo.

The DNA of eukaryotes is packaged with histone proteins innucleosomes, the repeating unit of chromatin (1). Althougheukaryotic genetic function takes place almost exclusivelywithin the context of this histone-DNA association, ourunderstanding of the dependency of genetic function onchromatin structure is largely qualitative. For example, nu-cleosomes residing at preferred positions within chromatinappear to be of central importance in the regulation of certaingenes, exemplified by yeast a-cell-specific promoters (2) andthe murine mammary tumor virus long terminal repeat (3).However, techniques for determining the positions of nucleo-somes with respect to DNA sequence have suffered fromsensitivity to artifacts and poor resolution, and this has led toconflicting results (4, 5), as well as to uncertainty in interpre-tation (6). These problems have been compounded by the factthat nucleosome position may be dynamic (7).

Ferrous-ion-chelating reagents tethered to DNA-bindingproteins have been used to cut DNA in the vicinity of themodification site through generation of hydroxyl radicals byusing the Fenton reaction (8). By modifying a recombinanthistone H4 molecule, we have developed a simple, base-pairresolution method for determining histone octamer position inany in vitro assembled nucleosomal complex. In this applica-tion of the method, we have mapped nucleosome core particles

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assembled on DNA from the Lytechinus variegatus 5S rRNAgene (9).

MATERIALS AND METHODS

Reagent Synthesis and Histone Derivatization. S-(tert-butyl)cysteamine formed from cysteamine and tert-butanol(10) was coupled to a 7-fold excess of EDTA by usingdicyclohexylcarbodiimide and N-ethyldiisopropylamine (11).Disulfide-linked 2-nitrophenylsulfenyl was exchanged for thetert-butyl (10) of the S-(tert-butyl)cysteaminyl EDTA to formS-(2-nitrophenylsulfenyl)cysteaminyl EDTA (EDTAcyst-NPS). The identity of the product, after purification by C18reverse-phase HPLC was confirmed by mass spectrometry,elemental analysis, and NMR. Mutant Xenopus laevis histoneoctamer containing histones H3(Cyst10 -> Ala), H4(Ser47Cys), wtH2A, and H2B(Ala7 -* Pro) expressed individually inbacteria and assembled into nucleosome core particles (NCP)(K.L., T. Rechsteiner, A.F., M. Waye, and T.J.R., unpublisheddata) was reacted with a 100-fold molar reagent excess in 50mM Tris*HCI, pH 7.4/2 M NaCl for 12 h at 25°C, and dialyzedagainst 5 mM potassium cacodylate, pH 6.0/2 M NaCl.Derivatized proteins were analyzed by Triton/urea/acetic acid(12) and SDS/PAGE (1:60 bis-acrylamide to acrylamide ra-tio). 63NiC12 staining and signal enhancement by using Amplify(Amersham) were as described by the manufacturer.DNA Fragments. Standard molecular biological methods

(13) were used unless otherwise noted. Asymmetric 146-bp 5SrDNA fragment (ASYM) containing bases -74 to +72 of theL. variegatus 5S rRNA gene (9), with coding strand bases -74and +71 mutated to A was prepared (14). Asymmetric 180-bp5S rDNA fragment (ASYM180) containing bases -88 to +89with ATC at its upstream end and base +89 changed to T wasa gift from T. Rechsteiner (Institut fur Molekularbiologie undBiophysik, ETH Zurich). Single-strand labeled ASYM wasprepared by denaturation in 10 mM NaOH, strand separationwith DEAE HPLC, alkaline phosphatase treatment, and la-beling with ['y-32P]ATP and polynucleotide kinase. DNAstrands were reannealed by heat denaturation and cooling. Apalindromic 146-bp 5S rDNA fragment (SYM) was preparedby ligating the insert from plasmid pST1, which contains sixligated monomers of the sequence from -68 to -2 of ASYMwith 5' and 3' extensions CTTGTCGAGATATC and CAA,respectively, into theEcoRV site of pST14, a pUC19 derivativecontaining an Xho I-EcoRV-Sal I linker substituted betweentheBamHI and HindlIl recognition sites of the polylinker. Theinsert was amplified by eight cycles of ligation of EcoRI-XhoI fragments into the EcoRI- and Sal I-digested vector, yielding40 EcoRV-releasable copies. The 84-bp EcoRV inserts wereisolated from pST5, 5' phosphates were removed by alkalinephosphatase, products were digested with Hindlll, and theresulting 73mer fragments were ligated to form SYM, which

Abbreviations: NCP, nucleosome core particle; ASYM, asymmetric146-bp 5S rDNA fragment; ASYM180, asymmetric 180-bp 5S rDNAfragment; SYM, palindromic 146-bp 5S rDNA fragment; EDTAcyst-NPS, S-(2-nitrophenylsulfenyl)cysteaminyl EDTA.*To whom reprint requests should be addressed.

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P,roc. Ai'd. Acad. Sci. USA 93 (1996) 1371

differs from bases 1-73 of ASYM only at base 6. SYM wasend-labeled as above without strand separation. ASYM non-coding strand "internally labeled" ladders were made byseparating Msp I-digested ASYM fragments, radioactivelylabeling the larger fragment as above, religating, digesting withEcoRV, and purifying by PAGE. ASYM coding strand andSYM internally-labeled ladders were made by digestingpTR30, which is analogous to pST5 but includes 17 extra basesat the 5' end of each 84-bp fragment (gift from T. Rechsteiner),with HindIII, labeling the 101-bp fragment as above, ligatingthese with an excess of nonphosphatased 73-bp EcoRV-Hindlll fragments from pST5, and then digesting with EcoRVand purifying as above. All base pairs were numbered relativeto the 5S transcript start site, which is assigned + 1. The 73-bpDNA fragment used to construct SYM extends from -74 to-2 by this numbering, and thus the 146-bp twofold symmetricligation product was numbered from -2 to -74 in bothdirections from its center.Assembly of Histone-DNA Complexes. Assembly reaction

mixtures containing 5 ,tM histone octamer and DNA fragmentwere dialyzed at 4°C against 20 mM Tris HCl, pH 7.4/1 mMEDTA in consecutive 2-h steps of 2 M, 0.85 M, 0.65 M, and 0.5M NaCl and finally overnight against 20 mM Tris HCl, pH 7.4.Precipitated material was removed by centrifugation at 4°C,and samples were either maintained at 4°C or incubated at37°C for 40 min. Nondenaturing PAGE was carried out in 16cm x 20 cm x 1 mm 5% (wt/vol) acrylamide (1:60 bis-acrylamide to acrylamide) gels containing 0.2x TBE (I x TBE= 90 mM Tris base/90 mM boric acid/2 mM EDTA, pH 8.0)at 4°C. Gels were prerun for 3 h at 250 V with bufferrecirculation and then run with samples loaded in 5% (wt/vol)sucrose under the same conditions. H3H4 tetramer was treatedidentically, except assemblies began with 1 M NaCl. Free metalions were removed from all materials (15).

Hydroxyl Radical Generation. Reaction of the nucleolyticsystem was carried at 4°C in a total volume of 80 ,lI with 0.4 ,uMNCP/37.5 mM Tris HCl, pH 7.4/0.5 ,uM (NH4)2Fe(SO4)2/3 mMascorbic acid/0.05% H202 for the times indicated. Reagents weredegassed and prechilled. Reactions were stopped by addition ofEDTA and thiourea, samples were precipitated and then ana-lyzed in denaturing gels (14) containing 25% (vol/vol) form-amide. Hydroxyl radical-free DNA ladders were generated byusing 100 ,uM (NH4)2Fe(SO4)2 and 200 ,uM EDTA.

RESULTSPreparation of a Nucleolytic Nucleosome Core Particle.

Potential sites for reagent attachment to the histone octamerwere determined by considering limited structural information(16). Using bacterial expression of histone proteins, we mu-tated Ser47 of histone H4 to cysteine, assembled this into H3H4tetramers and histone octamers, and derivatized the cysteinethiol with EDTAcyst-NPS (Fig. 1A). The adduct, EDTAcyst-H4, has the ability to bind heavy metal ions (Fig. 1B), andcauses reduced mobility of H4 in Triton/urea/acetic acid/polyacrylamide gels due to its negative charge and bulk (Fig.1C). Throughout this study, we have used histone mutantsH2B(Ala7 -> Pro), which imparts overall identity with thechicken sequence, and H3(Cysl" >- Ala) to avoid possiblecomplications with reagent modification of Cys' °. H3 Cys"0'will react with the reagent, but DNA cleavage in the subse-quent steps does not occur (A.F. and T.J.R., unpublisheddata). NCPs containing H3 Alal"0 behave identically to thewild-type H3 and to those assembled by using chicken histoneoctamer when analyzed by nondenaturing PAGE and x-raycrystallography (K.L., T. Rechsteiner, A.F., M. Waye, andT.J.R., unpublished data).The derivatized, recombinant histone octamer was assem-

bled into nucleolytic NCPs by using DNA fragments withsequences derived from the L. variegatus 5S rRNA gene

A NO2

COON ,C-NH-CH,-CH2-S-SCH ,C I2CK2\ /N -CH2-CH2-N

CH2 CH2COOH COOH

C Reagent - + -

H2A -__

B Reagent +

Oxidized 1-14 -

H3- _w

Oxidized H41- .

H3 >H2A/H2B -"

H4-, - _

1 2 3 4

H2B-

Derivatized H4--Underivatized H4 -

1 2 3

FIG. 1. Reagent derivatization of histone octamer. (A) Chemicalstructure of EDTAcyst-NPS. (B) Derivatization of H4 observed bySDS/PAGE in the absence of reductant. Coomassie staining (lanes 1and 2) and 63Ni2+/Amplify fluorography (lanes 3 and 4) of identicallyrun gels show derivatized (lanes 1 and 3) and underivatized (lanes 2and 4) histone octamer. (C) Derivatization of H4 observed by Triton/urea/acetic acid/PAGE. The Coomassie stained gel shows underiva-tized (lanes 1 and 3) and derivatized (lane 2) H4 Cys47-containinghistone octamer.

described in Materials and Methods. The ASYM NCP containsthe 5S rDNA fragment shown by nuclease digestion to yield a"positioned" nucleosome in vitro (17) and in vivo (18) andcorresponds to a defined sequence DNA used for crystallo-graphic studies of the NCP (14). The ASYM180 NCP containsthe sequence of ASYM with 17-bp native sequence extensionsof both DNA ends. The SYM NCP contains a perfectlypalindromic DNA sequence corresponding to the 5' half of theASYM coding strand. In addition to these NCPs formed withhistone octamer, H3H4 tetramer-DNA particles were pre-pared by using both ASYM and SYM. The 146-bp particles asobserved after electrophoresis on particle gels are shown inFig. 2A and Fig. 3A.

Cleavage Pattern Yields Accurate NCP Positions. Our ex-amination of the structural information for the NCP (16)suggested that the attachment of the EDTAcyst reagent to acysteine replacing H4 Ser47 would promote DNA cleavage viathe minor groove approximately 1-4 bp on either side of thedyad axis position at the DNA midpoint (Fig. 4C). The closecontact of the H4 protein to the minor groove on the inside ofthe particle would be expected to block cutting at sites over thenext half turn of DNA away from the dyad. For the reagent-modified ASYM NCP, the Fenton reaction resulted in onestrong DNA chain scission and weaker cuts 7 or 8 bases 3' ofthis site for each strand and NCP position (Fig. 2B, lanes 2 and3, positions -13 and +6; Fig. 2C, lanes 2 and 3, positions -9and + 10). All NCP positions cited in this work, as well asfurther studies using a variety of other sequences, show thatthis constellation of one strong and two weaker bands for eachstrand to be indicative of a single histone octamer position. Toprecisely identify the exact locations of cut sites, two types ofDNA ladders were prepared from the same DNA as wasincorporated into particles. The DNA for the "end-labeledladder" was labeled with 32p on the 5' terminus of one strand,and the DNA for the "internally labeled ladder" was labeledat a single site within the 5' half of the same strand. Limiteddigestion of internally labeled ladder free DNA by Fe2+/EDTA yielded a ladder of hydroxyl radical products visible

Biochemistry: Flaus et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

A ASYM _ SYM B ASYM coding strandA S~" U S, US ST

B UJ M S S M 1 T CG A M Lu s L("" u "), ;(s k 30' 910 90' 30' 90' 90 90' 0' 0'

1 2 34 56 -

-10-

c ASYM non-coding strand SYMU U U MS S MLT CG AM L. UU U MSS ML T' Cy A MI,0' 30' 90' 90 09090'90' 0r0I 30' 90' 90'30' 90 90'90' 0'

-10=- :.-_

-3-w

-1(1-

1 2 3 4 5, 6 7 X '1 10 11 2 3 14 1 2 3 4 5 6 7 81 1 ..1 4

._M .....

1) 10 12 13q 14

FIG. 2. NCPs assembled and mapped on 146-bp 5S rDNA sequences. (A) Nondenaturing PAGE of NCP assembled on ASYM 5' end-labeledon the coding strand (lanes 1 and 2) or the noncoding strand (lanes 3 and 4) and NCP assembled on SYM (lanes 5 and 6). Samples were maintainedat 4°C (lanes U, for unshifted) or heated at 37°C for 40 min in the absence of NaCl (lanes S, for shifted) before electrophoresis. The same positionsand shifting behavior occur for mapping in ionic strength conditions closer to physiological strength (200 mM NaCl; A.F., K.L., A. Maeder, andT.J.R., unpublished data). (B) Denaturing PAGE of the coding strand DNA from ASYM NCP after hydroxyl radical cleavage for unshifted (lanes1-3) and heat shifted (lanes 5 and 6) particles. Base-pair position calibration samples include a continuous ladder with undigested control (lanes4, 7, and 13; lanes M), an internally labeled ladder with undigested control (lanes 8 and 14; lanes L), and dideoxynucleotide sequence ladders (lanes9-12; lanes T, C, G, and A). Reaction times are in minutes. Bases are numbered relative to the first base of the 5S rRNA transcript. (C) DenaturingPAGE of ASYM noncoding strand DNA, labeled as in B. (D) Denaturing PAGE of NCP assembled on SYM, labeled as in B.

only for cuts 3' to the label-e.g., Fig. 2D, lane 8-allowingidentification of this base in the end-labeled ladder-e.g., Fig.2D, lane 7. The sites of single, strong chain scissions on theASYM coding strand were identified at bases -13 and +6 (Fig.2B, lanes 2 and 3) and on the ASYM noncoding strand at bases+10 and -9 (Fig. 2C, lanes 2 and 3). Since the NCP isapproximately twofold symmetric in the region of the DNAcenter (19), the symmetrically related cutting sites on comple-mentary strands indicate the position of the dyad symmetryaxis passing through the DNA, hence defining the nucleosomeposition. Considering the main cuts at -13 on the codingstrand and -9 on the noncoding strand and the second set withthe same separation at +6 and + 10, the symmetry of the NCPdictates that the dyad positions corresponding to the two setsmust be intermediate between cuts in a pair-i.e., at positions-11 and +8, respectively. Furthermore, the dyad must passapproximately either between two base pairs or through theplane of a base pair. The separation of 5 bp inclusive of themain cleavage sites shows that this DNA fragment is centeredon a single base pair at the dyad axis, with the main cutting siteon each strand two bases 5' of the dyad position (Fig. 4A). Theweak sites fall across the minor groove from the strong site onthe opposite strand, 4 to 5 bp from the dyad axis position (Fig.4C). These results demonstrate the accuracy of our nucleo-some mapping method and suggest that the canonical core

particle has an odd number of bases-i.e., 145 or 147.Heating Repositions the ASYM and SYM NCP. In addition

to the predominant -11 and +8 positions of ASYM NCP at4°C, two low occupancy positions with dyads at base pairs -2and -3 are detected in band intensity profiles (Fig. 2 B and C,lanes 2 and 3 and profiles not shown). Heating the mixture ofcomplexes to 37°C for 40 min transforms them entirely to thecentral positions -2 and -3 (Fig. 2 B and C, lanes 5 and 6).

SYM NCP has essentially only one position at base -11 (Fig.2D, lanes 2 and 3) and can be completely shifted to dyadpositions -2 and -3 by incubation at 37°C for 40 min (Fig. 2D,lanes 5 and 6; Fig. 3B). In this case, cuts on both sides of thedyad are superimposed on the single, symmetric sequencestrand (Fig. 4B). Alternatively, the cutting patterns of thecentrally located histone octamers are explained by a singledyad position between base pairs -2 and -3 for both ASYMand SYM NCPs. However, assignment of dyad positionsthrough bases is consistent with the single major band perstrand observed for the off-centered positions of the ASYMand SYM NCPs as well as other 146-bp particles that show onlyone position (A.F., unpublished data).The migration patterns produced by the various NCP sam-

ples by nondenaturing gel electrophoresis are explained bythese mapping results. SYM NCP migrates predominantly asa single band (Fig. 2A, lane 5), which is transformed to a fastermigrating species upon heating (Fig. 2A, lane 6), correspond-ing to a particle with DNA in primarily one off-centeredposition-i.e., -11-becoming a more compact particle withtwo superposed, central DNA positions-i.e., -2 and -3-after heating. ASYM NCP behaves similarly, showing threedistinct bands if maintained at 4°C (Fig. 2A, lanes 1 and 3) thatpresumably correspond to the DNA positions observed for thisparticle-i.e., -11, +8, and -2/-3. As a consequence ofheating, all ASYM NCP higher bands are converted to thelowermost band (Fig. 2A, lanes 2 and 4), which would corre-spond to the superposition of NCP with centrally positionedDNA-i.e., -2 and -3. The split upper band for ASYM NCPsuggests that the off-centered positions of DNA seen for thisparticle yield conformational differences that affect mobilitywithin the gel.

1372 Biochemistry: Flaus et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 1373

ASYM DNAA H3H4 tetramer NCP

0.8 1.0 1.5 2.0 1.0

mono-tetramercomplex

di-tetramercomplex

1 2 3 4 5

ASYM coding strandB T C G A 0.8 1.0 1.5 2.0 T C

0'30'90 0'30'900'30'90(0)'30'90'

octamert. ~11:complex WY 1

free %DNA

I4 t * ., :.'

w:!. W:

6..}* *

*o :.

123, 5 7I12 3 4 5 6 7 8

.''.~~~~~~~~. tw

9 10 I11 12) 13 14 15 1617 18

FIG. 3. Histone H3H4 tetramer-DNA complexes assembled and mapped on ASYM. All samples are unshifted. (A) Nondenaturingpolyacrylamide gel of H3H4 tetramer-ASYM complexes. Assembly was done at H3H4 tetramer to DNA ratios of 0.8, 1.0, 1.5, and 2.0 (lanes 1-4,respectively). NCP assembled on ASYM is included for comparison (lane 5). (B) Denaturing polyacrylamide gel of the coding strand DNA fromthe H3H4 tetramer-ASYM complex. The ratios are as forA at 0.8 (lanes 5-7), 1.0 (lanes 8-10), 1.5 (lanes 11-13), and 2.0 (lanes 14-16). Reactiontimes are in minutes. Bases are numbered as described for Fig. 2B. A calibrating dideoxynucleotide sequence ladder of the ASYM coding strandis also shown (lanes 1-4, 17 and 18).

Heating Has Little Repositioning Effect on ASYM180 NCP.Assembly and mapping of histone octamer on the 180-bp 5SrDNA fragment produces a pattern (Fig. 5, lanes 2 and 3)essentially identical to that observed for 146-bp ASYM NCP.There is no detectable occupancy of the central positions, asjudged by the absence of cutting at -4. The heightened cuttingat position -14, next to the main cut, but not at +5 comparedwith that observed when using the ASYM NCP probably stemsfrom the extended DNA of ASYM180 NCP near the dyadsubtly altering the diffusion of hydroxyl radical. Althoughthere is some increase in the occupancy of position -11relative to +8, heating of the 180-bp NCP for 40 min at 37°Cdoes not cause the substantial shift to the central positions asseen for the ASYM and SYM NCPs (Fig. 5, lanes 4 and 5).H3H4 Tetramer-DNA Complexes. Titration ofASYM DNA

with H3H4 tetramer reveals that a slower migrating species isgradually transformed to a faster migrating one as the ratio ofH3H4 tetramer to DNA is increased (Fig. 3A, lanes 1-4). Theintensity change with titration step in different regions of themapping pattern are linearly correlated with loss of H3H4tetramer binding at position -12/-11 and gain of binding atpositions -31 and +31 (Fig. 3B, lanes 5-16; Phosphorlmagerquantitation not shown). No differences were observed uponheating these complexes (A.F., unpublished data). Theseresults suggest that the slowest migrating band on the nonde-naturing gel is a monotetramer complex located almost en-tirely at position -12/-11, and the second fastest migratingband represents a dimer of H3H4 tetramers with individualtetramers positioned at base pairs -31 and +31. Other minorspecies also appear on the nondenaturing gel. Therefore,monotetramer appears to assemble almost solely at position-12/-11, consistent with previous findings (20, 21), but

tetramers take up completely new positions in a stackedassociation on the same face of ASYM in contrast to earliersuggestions (20, 22). The mobility of the particle, comparableto NCP, suggests that it is compact and that the DNA formsa continuous superhelix. In this case, and given that approxi-mately 80 bp of DNA are required to complete one full turnof superhelix around the H3H4 tetramer (19), the separationof 62 bp for the ditetramer complex indicates that the indi-vidual H3H4 tetramers are closely interacting and that theirmolecular dyad axes cannot be parallel. The angle of separa-tion of the two dyad axes around a common superhelix axiswould be approximately 81°-i.e., 62/80 = 0.775 of a full turn= +279 or equivalently -81°. Considering either of thestacked H3H4 tetramers, this arrangement would give H3 andH4 molecules at the interface the same orientation as H2A andH2B, respectively, within the NCP. This similarity of H3 andH4 with H2A and H2B agrees well with previous structuralresults (16, 23) and predicts that potentially useful reagentattachment sites may occur near the amino termini of all thehistone long a-helices.

DISCUSSIONThese results for NCP mapping at base-pair resolution illus-trate three important properties of nucleosome positioning.First, the shifting of histone octamer position on heating andthe rearrangement of H3H4 tetramer position because ofself-interaction point to the weakness of translational posi-tioning relative to the overall affinity of histone proteins forDNA. However, the underlying rotational position of thehistone unit is preserved throughout the various complexesthat we have investigated. Second, the monotetramer occupies

-+36

-+16

-+16

+6

:4._

--15

-25

.,_ --35p.8.

, 4pC..:

Biochemistry: Flaus et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

A -x -9 - 10 -11 -12 -13 -14 coding stranci3' 5'

5' 3non-codingY strand -X t9 -10 -11 12 -13 -14

B -6 5-n -4 32-3- -n - n -4 -5 6 -{) strand

3' 5

3 go,, gr

0:

0

5' 3'.-\Rstrand -6 -5 4 , .-) -5 -6

C

5'

3'

FIG. 4. Relationship ofDNA cutting sites to nucleosome position.(A) Schematic DNA backbone for unshifted ASYM NCP in the regionfrom -14 to -8 showing the main hydroxyl radical cleavage sites(filled ribose rings), hypothetical reagent locations (circled "r") andthe inferred dyad position (filled oval). (B) Schematic DNA backbonefor the heat-shifted SYM NCP in the region -6 to -2 showing thesuperposition of two positions on the dyad-symmetric DNA. Thecenter of the DNA is indicated by an open oval. (C) Model of B-formDNA showing main (black) and subsidiary (gray) ribose rings cleavedas viewed from inside the NCP (Upper) and down its superhelix axis(Lower). The molecular dyad axis is indicated by a filled oval.

a position consistent with histone octamer locations observedby others on larger DNA fragments (18, 24, 25). We confirmat high resolution the suggestion that the H3H4 tetramer is a

major determinant of in vitro nucleosome positioning (20, 21,26). Third, the occurrence on ASYM of two histone octamerpositions but only one significant H3H4 tetramer positionindicates that the H2AH2B dimer and the DNA-terminalregions can also play a role in positioning on 146-bp lengthDNA.

Previously, measurement of relative histone octamer-DNAaffinities by competitive exchange of histones between DNAfragments of differing sequence (27) has been facilitated byattaining binding equilibrium at salt concentrations severaltimes higher than physiological. At low ionic strength in theabsence of other factors, the association of histones with DNAis irreversible: interfragment exchange is not observed (28).The interpretation of interfragment exchange data at elevatedsalt concentrations encounters complications, however, sincethe details of histone-DNA interaction may not be equivalentat high and low ionic strengths (29). Our observation ofposition shifting by heating reveals that a different balance ofinteractions does occur within the NCP at high and low ionicstrengths, at least on 146-bp DNA fragments. We interpret thecentering of histone octamer on 146-bp DNA when heated at

ASYM 180 coding strandL U U S S A GcTrO' 3 901 30 90

q

-+13

* g 6

06

4wi

p=

.i._gpw.........S.".. 'S'-'13.,;:XX'. v e:

'">' :''' '3U'.

:' U-

Ip

1 2 3 4 5 6 7 8 9

FIG. 5. NCPs assembled and mapped on 180-bp 5S rDNA. Dena-turing PAGE of the coding strand DNA from the ASYM180 NCP afterhydroxyl radical cleavage for unshifted (lanes 1-3; lanes U) andheat-shifted (lanes 4 and 5; lanes S) particles shows the same cuttingsites as for the ASYM NCP (Fig. 2B). The arbitrary dideoxy-nucleotide sequence ladder shown was calibrated by comparison toequivalent length stops in the ladder of Fig. 2. Bases are numbered asdescribed for Fig. 2B.

low salt concentration to mean that the most stable particlesunder these conditions have maximized the number of ionicinteractions between protein and DNA. In support of thisconclusion, position shifting to the central location did notoccur on the longer, 180-bp 5S rDNA sequence. We note thatrearrangement has been observed on longer fragments of the5S rDNA sequence trapped within polyacrylamide gels andthat additional sequences may favor additional sites (7).Our demonstration of NCP position shifting in solution at

low ionic strength suggests that the alternative approach ofintrafragment equilibration for the study of chromatin ener-getics may be feasible. Although limited here to DNA 146 bpin length, we have observed position shifting for longer DNAfragments of other sequences by using the conditions described(A.F., unpublished data). The mapping method introducedhere allows accurate assignment ofbinding sites and estimationof their relative occupancies via titration experiments. More-over, position shifting is a dynamic process, and assuming atemperature independence of nucleosome and DNA structure,the kinetic properties of histone octamer mobility betweenindividual positions may be measurable (30).Understanding chromatin function depends on the accurate

assignment of nucleosome positions whether they are uniqueor multiply overlapping. The moderate energy barrier tohistone octamer translation demonstrates that significant re-arrangements of nucleosomes can occur under physiologicallyrelevant conditions and is apparently relevant to variability innucleosome positioning observed in vivo (31). In this respect,some mechanisms for transcriptional repression, initiation,and elongation which depend on chromatin remodeling mayrely on the ability of the nucleosome to shift over one or moreturns of the DNA duplex (32-34). Indeed, the effect ofacetylation upon TFIIIA access to the internal control region

.. I_ _ ... .... == .;

1374 Biochemistry: Flaus et al.

p-51

3'

Proc. Natl. Acad. Sci. USA 93 (1996) 1375

(ICR) of the Xenopus borealis somatic 5S rRNA gene (35)could be caused by increased mobility of the acetylatednucleosome, making important sections of the ICR transientlyavailable to the transcription factor.The nucleosome mapping method reported here can be

performed on the picomole scale in vitro and is applicable toany DNA sequence. Analogous methods may permit mappingin chromatin-assembly nuclear extracts and isolated nuclei.Single-base-pair resolution mapping and temperature-inducedposition equilibration make possible the investigation of themechanism and energetics of nucleosome positioning underphysiologically relevant conditions.

We thank A. Mader and T. Rechsteiner for discussion and, togetherwith R. Richmond, for helping to purify recombinant histones andDNA. This work was supported by the Swiss National Fund forScience.

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R. T. (1992) Genes Dev. 6, 411-425.3. Richard-Foy, H. & Hager, G. L. (1987) EMBO J. 6, 2321-2328.4. Pinla, B., Barettino, D., Truss, M. & Beato, M. (1990)J. Mol. Biol.

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