Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex...

doi:10.1006/jmbi.2000.4069 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 302, 315±326

Differential Remodeling of the HIV-1 Nucleosomeupon Transcription Activators and SWI/SNFComplex Binding

Dimitar Angelov1, Monique Charra1, Michel Seve2, Jacques CoÃ teÂ 3

Saadi Khochbin4 and Stefan Dimitrov1*

*Corresponding author

Permanent address: D. Angelov, Institute of SolidState Physics, Bulgarian Academy of Sciences, 1784So®a, Bulgaria.

Abbreviations used: EMSA, electrophoretic mobility-shift analysis; HIV-1, human immunode®ciency virustype 1; LTR, long terminal repeat; TPA, tetradecanoylphorbol acetate; TNF-a, tumor necrosis factor type a;TPX, trapoxin; TSA, trichostatin A; DHS, DNase Ihypersensitivity sites; CHIP, chromatinimmunoprecipitation.

E-mail address of the corresponding author:[email protected]

0022-2836/00/020315±12 $35.00/0

Introduction

In eukaryotic cells, DNA is packaged into chro-matin. The basic chromatin subunit is the nucleo-some, which consists of a fragment of DNA ofabout 200 bp wrapped around an octamer of corehistones, two each of H2A, H2B, H3 and H4. Boththe histone octamer and the nucleosome have beencrystallized and their structure solved by X-ray-crystallography (Arents et al., 1991; Luger et al.,1997). The crystallographic data reveal that the his-tone octamer is tripartite, with a centrally located(H3-H4)2 tetramer ¯anked by two H2A-H2Bdimers (Arents et al., 1991). All four types ofhistones within the octamer possess a commonstructural motif, the histone fold, and ¯exible

1Laboratoire de BiologieMoleÂculaire et Cellulaire de laDiffeÂrenciation, eÂquipeMeÂcanismes d'Assemblage duMateÂriel GeÂneÂtique, INSERMU 309, Institut Albert BonniotDomaine de la Merci, 38706 LaTronche Cedex, France2Laboratoire de Biologie duStress Oxydant (LBSO), UJFDomaine de la Merci, 38700 LaTronche Cedex, France3Laval University CancerResearch Center, Hotel-Dieu deQuebec, 9 rue McMahonQuebec, Qc G1R-2J6, Canada4Laboratoire de BiologieMoleÂculaire et Cellulaire de laDiffeÂrenciation, eÂquipeStructure de la chromatine etexpression des geÁnes, INSERMU 309, Institut Albert BonniotDomaine de la Merci, 38706 LaTronche Cedex, France

Here we have examined HIV-1 nucleosome remodeling upon the bindingof transcription factors and the SWI/SNF complex using a novelapproach. The approach combines UV laser protein-DNA crosslinking,electrophoretic mobility-shift analysis and DNase I protection analysiswith immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histoneNH2 tail-DNA interactions. However, the simultaneous binding of thetranscription activators Sp1, NF-kB1, LEF-1 and USF synergisticallyincreased the release of histone NH2 tails from nucleosomal DNA. In con-trast, the binding of SWI/SNF complex to HIV-1 nucleosome disruptedstructured histone domain-DNA contacts, but not histone NH2 tail-DNAinteractions. Stable remodeled nucleosomes, (obtained after detachmentof SWI/SNF), displayed identical structural alterations with those boundto SWI/SNF. These results demonstrate a different in vitro remodeling ofthe HIV-1 nucleosome upon the binding of multiple transcription activa-tors and of SWI/SNF complex.

# 2000 Academic Press

Keywords: nucleosome; histone tails; remodeling; activators; SWI/SNFcomplex

# 2000 Academic Press

http://www.idealibrary.com

mailto:[email protected]

316 SWI/SNF and Activator-nucleosome Remodeling

unstructured NH2 termini. The histone NH2 tailsare located external to the nucleosome and are thesubject of different post-translational modi®cationssuch as acetylation and phosphorylation, andthus they have to interact directly with chromatin-modifying histone acetyltransfrerase and deacety-lase complexes as well as with kinases (reviewedby Wade et al., 1997; Wolffe & Pruss, 1996; Roth &Allis, 1996; Luger & Richmond, 1998). In addition,repressor complexes such as the Ssn6-Tup1 com-plex (Edmonson et al., 1996) and the SIR complexin Saccharomyces cerevisae (Hecht et al., 1995) arebelieved to physically associate with nucleosomesby binding the histone tails. Moreover, the bindingof Ssn6-Tup1 to the tails is negatively regulated byhistone acetylation. In addition, transcription fac-tors seem to exhibit enhanced af®nity for taillessnucleosomes or nucleosomes containing hyperace-tylated histones, suggesting that the tails mayregulate factor access to their cognate DNAsequences (Lee et al., 1993; Vettese-Dadey et al.,1996; Mutskov et al., 1998).

Some of the chromatin remodeling complexesmay act also through the histone tails. Forexample, the interaction between the Drosophilaremodeling complex NURF and nucleosomes isimpaired by trypsin removal of the NH2 histonetermini, and its ATPase activity is inhibited byeach of the histone tails (Georgel et al., 1997). SinceNURF probably remodels nucleosomes by ATP-dependent histone octamer sliding (Hamiche et al.,1999), the possible interaction between the histoneNH2 tails and NURF could play an important rolein its action.

Another remodeling complex, SWI/SNF, ef®-ciently perturbs both native and tailless nucleo-somes (Guyon et al., 1999). In vivo, this complex isrequired for the maintenance of transcription ofdifferent genes (Winston & Carlson, 1992;Sudarsanam et al., 1999). The SWI/SNF proteinsare integral components of the yeast RNA poly-merase II holoenzyme and thus provide theholoenzyme with the capacity to disrupt promoternucleosomal organization (Wilson et al., 1996). Thenucleosome-remodeling mechanisms of SWI/SNFand of its related RSC complex are not reallyknown and are currently intensively studied(Lorch et al., 1998; CoÃ teÂ et al., 1998; Schnitzler et al.,1998; Lee et al., 1999; Bazett-Jones et al., 1999).SWI/SNF acts transiently in remodeling thenucleosome structure by directly binding tonucleosomes. It uses ATP hydrolysis to disrupt his-tone-DNA contacts, and as a result, an enhancedsensitivity of nucleosomal DNA to digestion withDNase I and restriction enzymes is observed. Fur-thermore, the SWI/SNF complex creates loopdomains in DNA and polynucleosome arrays, andthe altered nucleosome state persists for severalhours after detachment from the nucleosome.

The human immunode®ciency virus type 1(HIV-1) long terminal repeat (50LTR) is a very suit-able system for investigating mechanisms ofnucleosome remodeling since the chromatin struc-

ture of the integrated HIV-1 has been relativelywell characterized (Verdin, 1991; Verdin et al.,1993; van Lint et al., 1996). In addition, in vitrochromatin assembly of the HIV-1 50LTR has beenreported (Widlak et al., 1997; Sheridan et al., 1997;Steger et al., 1998). In vivo, ®ve nucleosomes (nuc-0to nuc-4) have been shown to be precisely posi-tioned in the 50LTR and de®ne two nucleosome-free regions spanning nucleotides ÿ232 to �19 and179 to 289, respectively. A nucleosome (nuc-1),localized between these two regions, can be dis-rupted upon treatment with tetradecanoyl phorbolacetate (TPA) and tumor necrosis factor type a(TNF-a; Verdin et al., 1993), or with the speci®c his-tone deacetylase inhibitors trapoxin (TPX) or tri-chostatin A (TSA). The disruption of nuc-1 and thepossible histone displacement were a-amanitininsensitive and independent of transcription,suggesting that this chromatin remodeling is a pre-requisite for transcription. In addition, it has beenshown that the region between nuc-0 (nucleotidesÿ392 to ÿ232) and nuc-1 (nucleotides 21 to 165) isresistant to microccocal nuclease digestion (Verdinet al., 1993). Nevertheless, this region, large enoughto accommodate a nucleosome, was suggested tobe nucleosome free since it contains the bindingsites of numerous transcription factors (USF, ETS,LEF-1, NF-kB, Sp1, etc.) and more importantly,two DNase I hypersensitivity sites (DHSs)observed upon digestion with DNase I of nucleiisolated from HIV-1 persistently infected cells(Verdin et al., 1993). This hypothesis, however, wasquestioned in a series of in vitro experiments bySteger & Workman (1997). These authors havedemonstrated that the transcription factors USF,ETS, LEF-1, NF-kB and Sp1 can gain access to theirbinding sites within a nucleosome core reconsti-tuted on a HIV-1 150 bp fragment localizedbetween nuc-0 and nuc-1 and encompassingnucleotides ÿ199 to ÿ49 of the 50LTR region. Fur-thermore, they found that when the same 150 bpfragment was incorporated into a nucleosomalarray, the binding of Sp1 and NF-kB inducedregions of enhanced DNase I sensitivity speci®c forthe HIV-1 nucleosome. These DNase I-enhancedsensitivity regions were shown to be similar to theDHSs observed in isolated nuclei, but still the HIV-1 nucleosome was found to remain intact even inthe presence of the chromatin-remodeling complexSWI/SNF or the histone chaperone nucleoplasmin.These data suggest that the constitutive DHSsobserved in vivo may re¯ect the presence of a tern-ary complex, consisting of DNA, histones and tran-scription factors. Therefore, there is a disagreementbetween the in vitro results (Steger & Workman,1997) and the interpretation of the in vivo nuclease-digestion data (Verdin et al., 1993; van Lint et al.,1996) on the chromatin organization of the HIV-150LTR region.

Our long-term goal is to understand the role ofchromatin structure in the control of HIV-1 tran-scription regulation. The results described here rep-resent a ®rst step towards this goal. We have

SWI/SNF and Activator-nucleosome Remodeling 317

analyzed the effect of transcription activators andSWI/SNF binding to a nucleosome reconstitutedon a HIV-1 50LTR fragment, containing the bindingsites for Sp1, NF-kB, LEF-1 and USF, by using anovel combination of UV laser protein-DNA cross-linking, electrophoretic mobility-shift analysis(EMSA) and DNase I footprinting. UV laserirradiation induces crosslinking only via the non-structured histone NH2 tails, and thus presents aunique tool for studying histone tail interactionswith nucleosomal DNA. Comparison of the cross-linking data with the DNase I digestion pattern oftranscription activator-bound and SWI/SNF-remo-deled HIV-1 nucleosomes allowed us to show thatdifferent alterations in nucleosome structure wereinduced by these remodeling agents.

Results

Prior to analysis of transcription factor-inducedHIV-1 nucleosome remodeling, we ®rst veri®edthat puri®ed transcription factors could bind theircognate sites on in vitro reconstituted nucleosomeson a 150 bp DNA fragment derived from 50LTRHIV-1, containing binding sites for USF, LEF-1,NF-kB and Sp1 (Figure 1(a)). Electrophoretic mobi-lity-shift analysis demonstrates ef®cient nucleo-some reconstitution by the octamer-transfermethod (a quantitative estimation shows that morethan 85 % of 32P-labeled DNA was nucleosomereconstituted; Figure 1(b)). Subsequent bindingassays with these HIV-1 nucleosomes and the tran-scription factors USF, LEF-1, NF-kB and Sp1showed shifted complexes in all cases (Figure 1(b)).USF and LEF-1 formed one major complex, whileNF-kB and Sp1 formed two and three, respectively.This is consistent with the fact that the HIV-150LTR DNA fragment used for reconstitution con-tains one recognition site for USF and LEF-1, twofor NF-kB and three for Sp1. The multiple tran-scription factors can also bind simultaneously tothe reconstituted nucleosome (Figure 1(b); see alsoFigure 5). These results therefore con®rm the dataof Steger and Workman (1997), demonstrating thattranscription factors can invade the HIV-1 nucleo-some.

The simultaneous binding of multipletranscription factors to the HIV-1 nucleosomesynergistically affects the release of the NH2

tails from nucleosomal DNA

Once the results of Steger and Workman (1997)had been con®rmed we investigated how the bind-ing of transcription factors affects HIV-1 nucleo-some structure. We were particularly interested inthe fate of histone NH2 tail-nucleosomal DNAinteractions upon the binding of transcription fac-tors. For this we used a novel UV laser inducedprotein-DNA crosslinking approach (Moss et al.,1997; Mutskov et al., 1998).

Irradiation of protein-DNA complexes with highintensity UV lasers, in contrast to that with conven-

tional UV light sources, induces ef®cient protein-DNA crosslinking (Hockensmith et al., 1986;Angelov et al., 1988; Pashev et al., 1991;Hockensmith et al., 1993a,b; Stefanovsky et al.,1989). This high ef®ciency is achieved by thebiphotonic mechanism of the crosslinking operat-ing in the case of laser irradiation (Angelov et al.,1988; Pashev et al., 1991; Hockensmith et al., 1991).A peculiarity of the laser methodology is that corehistone-DNA crosslinking is achieved exclusivelyvia the NH2 tails both in chromatin and in isolatednative or reconstituted nucleosomes (Stefanovskyet al., 1989; Mutskov et al., 1998).

Indeed, UV laser irradiation of tailless nucleo-somes (obtained after protease digestion) did notinduce histone-DNA crosslinking. In addition, adramatic decrease in crosslinking ef®ciency wasobserved in the interval 0.2-0.6 M NaCl (in thisinterval of NaCl concentration the histone tails areremoved from their interactions with nucleosomalDNA), further con®rming the above conclusion.Crosslinking ef®ciency, therefore, directly re¯ectsthe extent of histone tail-DNA contact, whichmakes UV laser crosslinking a particularly power-ful method for studying this type of interaction(Pashev et al., 1991; Stefanovsky et al., 1989;Mutskov et al., 1998).

The experimental strategy (laser chromatinimmunoprecipitation (CHIP)) we have used forstudying the effect of nucleosome-bound transcrip-tion factors on histone NH2 tails-DNA crosslinkingef®ciency is shown in Figure 2. Brie¯y, 32P-labeledparticles, containing one or multiple bound tran-scription factors, were laser-irradiated with identi-cal UV doses and the covalent core histone-DNAcomplexes were immunoprecipitated using highlyspeci®c antibodies against individual core histones.Stringent immunoprecipitation conditions wereused to ensure the complete removal of the non-crosslinked proteins from nucleosomal DNA (seeMaterials and Methods). Mesurement of theamount of individual immunoprecipitated histo-ne-32P-labeled DNA covalent complexes allowedthe ef®ciency of histone NH2 tail-DNA crosslinkingto be estimated.

The antibodies against the core histones H2A,H2B and H4 are highly speci®c: no cross-reactionwith other proteins is observed (Mutskov et al.,1998), and they have previously been successfullyused for the precipitation of covalent histone-DNAcomplexes induced by laser irradiation of nucleiand of isolated chromatin and nucleosomes(Dimitrov et al., 1990, 1992; Mutskov et al., 1998).

The immunoprecipitation data are presented inFigure 3. As can be seen, only in the case of threebound Sp1 is the ef®ciency of crosslinking of H2A,H2B and H4 histone NH2 tails decreased, but thisdecrease is very small and does not exceed 10-15 %for the different histone tails. However, when allfour factors were bound to the HIV-1 nucleosome,a signi®cant decrease (60-65 %) of crosslinking ef®-ciency for all three histone tails is observed. Thus,the binding of all factors on HIV-1 nucleosomes

Figure 1. Transcription factors are able to bind to HIV-1 reconstituted nucleosomes. (a) Schematic presentation ofthe 150 bp HIV-1 50LTR fragment used for nucleosome reconstitution. The locations of the factor binding sites areshown. (b) EMSA of transcription factor bound to naked DNA and reconstituted nucleosomes (Nuc). The concen-tration of the different transcription factors used for the binding studies are given. The arrows show the positions ofDNA and of nucleosomes.


releases a signi®cant amount of the histone NH2

tails from their interactions with DNA. However,the various transcription factors, when bound sep-arately on HIV-1 nucleosomes, exhibited only avery weak effect on the histone tail-DNA inter-actions. If one assumes that the bound multipletranscription factors will affect additively histonetail-DNA interaction, the calculated additive effectupon their binding should be negligibly weak,since the crosslinking ef®ciency of each individualhistone tail for the multiple factors-bound nucleo-some is essentially the same as that of the control,i.e. nucleosome not containing bound transcriptionfactors (Figure 3, �calculated). In other words, uponan assumption of an additive effect we should notobserve a diminution of the crosslinking histonetail ef®ciency for multiple factors-bound nucleo-some. Since this is not the case (a signi®cantdecrease of the histone tail-DNA crosslinking wasobserved) we interpret the immunoprecipitationdata to demonstrate a synergistic effect of tran-scription factors binding on the histone NH2 tailsrelease from their interaction with HIV-1 nucleoso-mal DNA.

Histone H3-DNA interaction within these tran-scription factor-bound HIV-1 nucleosomes couldnot be investigated. The antibodies we have raisedagainst histone H3, although highly speci®c, werefound not to work in the high salt and detergentconcentration conditions used for immunoprecipi-tation.

The binding of SWI/SNF to the HIV-1nucleosome disrupts the interactions of thehistone-structured domains with DNA

We have demonstrated that the binding of mul-tiple transcription factors to HIV-1 nucleosomeresulted in a signi®cant release of the histone NH2

tails from their interaction with DNA. However, incell nuclei, chromatin remodeling is also stronglydependent on high molecular mass remodelingcomplexes such as SWI/SNF, NURF, CHRAC,RSC, ACF, etc. (reviewed by Kornberg & Lorch,1999; Travers, 1999; Pollard & Peterson, 1998). Toact, these complexes require ATP hydrolysis, butthe mechanism of nucleosome disruption is farfrom clear. We therefore investigated how the

Figure 2. Schematic represen-tation of the immunoprecipitationprocedure (laser CHIP) used forstudying the effect of transcriptionfactors and SWI/SNF binding onthe histone NH2 tail-DNA inter-actions.


SWI/SNF complex remodels HIV-1 nucleosomesusing EMSA, a DNase I-protection assay and UVlaser protein-DNA crosslinking.

Incubation of both naked HIV-1 DNA or recon-stituted nucleosomes with a suf®cient amount ofSWI/SNF results in complete binding as judged byEMSA (Figure 4, lanes 2 and 4). Since we haveused standard polyacrylamide gels, SWI/SNF-DNA and SWI/SNF-nucleosome complexes havenot entered substantially the gel, which is in agree-ment with the results of CoÃ teÂ et al., 1998. The bind-ing of SWI/SNF to both DNA and HIV-1nucleosome was independent of ATP (Figure 5 anddata not shown). Interestingly, in the absence ofATP, the DNase I footprinting analysis of SWI/SNF-bound nucleosomes showed weak, but repro-ducible, protection in two regions overlapping theUSF and NF-kB binding sites (Figure 5, comparelanes 1-4). This protection was observed at differ-ent concentrations of SWI/SNF (data not shown).

Since this pattern is not observed in SWI/SNF-bound naked DNA (Figure 5 , lanes 5 and 6), weattributed the protection to a speci®c binding ofSWI/SNF to these regions of the HIV-1 nucleo-some. The addition of ATP and MgCl2, requiredfor the activity of the SWI/SNF complex, results indramatic changes in the DNase I cutting pattern(Figure 5, lanes 7 and 8), re¯ecting the perturbationof histone-DNA interactions in the SWI/SNF-bound nucleosome (CoÃ teÂ et al., 1994, 1998;Imbalzano et al., 1996; Guyon et al., 1999).

Does this phenomenon, as in the case of tran-scription factor-nucleosome binding, involve per-turbations in the interactions of histone NH2 tailswith nucleosomal DNA? To answer this questionwe performed laser CHIP experiments on SWI/SNF-bound HIV-1 nucleosomes (for details of theexperimental strategy, see Figure 2). As demon-strated (Figure 6(a)), the ef®ciency of immunopreci-pitation of each individual covalent histone-DNA

Figure 3. Binding of the fourtranscription factors to HIV-1reconstituted nucleosomes synergis-tically affects the factor-inducedrelease of the histone NH2-tails.Transcription factors were allowedto interact with the 32P-labeled160 bp nucleosome particles underoptimal conditions for saturation oftheir cognate sequences on thereconstituted particles. Particles,either not containing (control) orcontaining three Sp1, or two NF-KB1, or one LEF-1 or one USF, orall these factors (�), were laser irra-diated with identical doses and theindividual covalently linked his-tone-DNA complexes were immu-noprecipitated by using speci®c

antibodies against histones H2A, H2B and H4 . The amount of DNA within the immunoprecipitated complexes wasdetermined by Cerenkov counting. Histograms showing the percentage of immunoprecipitated individual histone-DNA complexes in the presence of bound transcription factors relative to that in the absence of bound ones are pre-sented. The amount of immunoprecipitated DNA from the control irradiated nucleosomes (not containing boundtranscription factors) is taken as 100 %. Calculated � represents the amount of immunoprecipitated individual com-plexes, calculated by assuming an additive effect of the bound transcription factors on the release of NH2-histonetails (see Materials and Methods). In this calculation the experimental data for the effect of each factor (this Figure)were used.


complex is essentially the same as for the controlwhich was carried out on HIV-1 nucleosomes irra-diated in the absence of SWI/SNF. In addition, thesame results were obtained for samples not con-taining ATP and MgCl2 (Figure 6(b)). Since the ef®-ciency of crosslinking is a direct measure of histoneNH2 tail-DNA contacts, we can conclude that thebinding of SWI/SNF to HIV-1 nucleosome doesnot involve changes in the amount of these con-tacts. Thus, the observed SWI/SNF induced per-turbation of HIV-1 nucleosome is due todisruptions of the histone-structured domain DNAinteractions.

The SWI/SNF stable remodeled HIV-1nucleosomes exhibit the same amount ofhistone NH2 tails-DNA contacts asnative nucleosomes

SWI/SNF and its related RSC chromatin-remo-deling complexes, upon binding, induce a stableremodeling of the nucleosome structure, whichpersists for several hours after their detachment(CoÃ teÂ et al., 1998; Lorch et al., 1998; Schnitzler et al.,1998; Guyon et al., 1999). The stable altered nucleo-somes observed after detachment of the remodel-ing complex seemed to retain the features of theremodeled nucleosomes found in the presence ofbound SWI/SNF or RSC. If this is really the case,within this stable altered nucleosome the histonetails-DNA interactions should not be affected, i.e.only the contacts between the histone-structureddomains and DNA should be altered. To checkthis, we ®rst generated stable remodeled nucleo-somes by competing the SWI/SNF-bound HIV-1labeled nucleosomes with a high excess of

unlabeled nucleosomes (Figure 4, lanes 5 and 6). Inagreement with the data in the literature (CoÃ teÂet al., 1998; Schnitzler et al., 1998; Guyon et al.,1999), these nucleosomes retained an alteredDNase I pattern characteristic of the SWI/SNF-con-taining nucleosomes (Figure 5, lane 10). It shouldbe noted that in the absence of ATP and MgCl2,the DNase I cutting pattern of the competednucleosomes was identical with the control, non-competed nucleosomes (Figure 5, compare lanes 1,3 and 4). The stable remodeled and the controlnucleosomes were irradiated at identical doses andwere further subjected to laser CHIP. As seen inFigure 6(a) the amount of immunoprecipitatedindividual histone-DNA complexes for both con-trol and stable remodeled nucleosomes is the same,and it is identical with that of precipitated histone-DNA complexes for SWI/SNF-bound and controlnucleosomes irradiated in the absence of ATP andMgCl2 (Figure 6(b)). Therefore, the amount of his-tone NH2 tails-DNA contacts are preserved in thestable remodeled nucleosomes.

Discussion

The binding of multiple transcription activatorsremodels the HIV-1 nucleosome: the majorityof histone NH2-tails are released from theirinteraction with nucleosomal DNA

Here, we have studied the effects of transcriptionfactor and SWI/SNF binding on HIV-1 nucleo-somes in vitro. In agreement with the literature(Steger & Workman, 1997) we have found thatSp1, NF-kB1, LEF-1 and USF can invade the HIV-1nucleosome (Figure 1(b)). The UV laser protein-

Figure 4. EMSA of DNA and reconstituted nucleo-somes containing bound SWI/SNF complex with andwithout competitor nucleosomes. Reconstituted nucleo-somes were supplemented with 0.5 mM ATP and 4 mMMgCl2, SWI/SNF (10 nM ®nal concentration) wasadded to the designated samples and the reaction mix-tures were incubated for 45 minutes at 30 �C. Once thereactions completed, the designated samples were com-peted with 30 times excess of unlabeled linker histone-depleted oligosomes to remove bound SWI/SNF. Sub-sequently all reactions were analyzed by EMSA.

Figure 5. DNase I footprinting of SWI/SNF remo-deled nucleosomes in (a) the absence or (b) the presenceof ATP. The binding conditions are identical with thoseof Figure 4. The binding sites of SWI/SNF are desig-nated by the continuous lines on the left. The numberson the right refer to the nucleotide position relative tothe start transcription site.


DNA crosslinking CHIP revealed that the bindingof individual factors to the HIV-1 nucleosome doesnot affect the interaction of the histone NH2 tailswith DNA. However, the simultaneous binding ofmultiple factors (Sp1, NF-kB1, LEF-1 and USF) tonucleosomes could dramatically alter histonetail-DNA interactions; up to 60 % of the tails werereleased from their interactions with DNA in theseconditions. This is suggestive of a synergistic effecton histone NH2 tail release upon binding of mul-tiple transcription activators to HIV-1 nucleosome.This synergistic effect might simply re¯ect a com-petition between histone tails and transcription fac-tors for DNA sites. When a factor binds DNA itmay locally disrupt histone tail-DNA contacts andrelease some of the tails from DNA. Alternatively,the tails, being quite ¯exible (Cary et al., 1978; vanHolde, 1988), may interact de novo with someneighboring DNA sequences, in which case nodifference in the ef®ciency of histone tail cross-linking would be predicted (the ef®ciency of cross-linking re¯ects the amount of tails in close contactwith the DNA, but not their localization on theDNA). However, when multiple activators withclosely positioned cognate sequences are bound tonucleosomal DNA, the tails may no longer be ableto interact with DNA because of steric reasons, i.e.

no neighboring DNA sites are available since theyare already occupied by other activators. This is inagreement with data in the literature whichdemonstrate an enhanced af®nity of transcriptionfactors for tailless nucleosomes (Lee et al., 1993).Thus, the binding of mutiple activators to the HIV-1 nucleosome remodels the nucleosome by releas-ing more than half of the amount of histone NH2

tails from their interactions with nucleosomalDNA.

SWI/SNF remodeling of the HIV-1 nucleosomedoes not involve changes in the amount ofhistone NH2 tail-DNA contacts

The SWI/SNF complex has been shown to par-ticipate in the remodeling of chromatin for tran-scription by disrupting histone-DNA interactionsand allowing easier access of activators to nucleo-somal DNA (reviewed by Peterson & Tamkun,1995; Pollard & Peterson, 1998; Pazin & Kadonaga,1997). SWI/SNF nucleosome disruption is ATP

Figure 6. Binding of SWI/SNF does not affect histoneNH2 tail-DNA interactions. (a) 32P-labeled 160 bp DNAparticles were incubated with SWI/SNF (10 nM ) at30 �C for 45 minutes in a binding buffer containing0.5 mM ATP and 4 mM MgCl2. Upon completion of thereactions the designated samples were passed throughcompetition with unlabeled histone linker depleted oli-gosomes. All samples were irradiated at identical dosesand the individual covalent histone-DNA complexeswere precipitated with speci®c antibodies and theamount of precipitated DNA within the complexes wasmeasured by Cerenkov counting. The data are presentedas histograms showing the percentage of immunopreci-pitated individual histone-DNA complexes relative tothat of the control (non-SWI/SNF treated) nucleosomes.The amount of immunoprecipitated DNA from the con-trol irradiated nucleosomes is taken as 100 %. (b) Sameas (a), but instead of ATP, 0.5 mM dCTP was added.The binding buffer did not contain MgCl2.


dependent, but the mechanism of disruption is notwell understood. Indeed, the binding of SWI/SNFto a nucleosome changes the DNase I cutting pat-tern along the entire nucleosomal DNA length, thiscutting pattern being preserved even in the stablealtered nucleosomes several hours after its detach-

ment. At the same time histones are found associ-ated with DNA in remodeled nucleosomes. Thus,we have a paradoxical situation: nucleosomalDNA behaves like naked DNA, but is still associ-ated with histones (Kornberg & Lorch, 1999). Howmay this happen? Our histone crosslinking datashed light on this peculiar nucleosome organiz-ation. We found that the amount of histone NH2

tail-DNA contacts is quantitatively preserved inboth SWI/SNF-bound and stable remodelednucleosomes following detachment of the remodel-ing complex. Thus, the altered DNase I cutting pat-tern and the exposure of restriction enzymecleavage sites should re¯ect disrupted histonestructured domain-DNA interactions only. There-fore, within the SWI/SNF remodeled nucleosomewe could imagine the histone structured domainsto be at least partially released from their inter-action with nucleosomal DNA, while the histoneNH2 tails remain stably attached to the DNA, andthus anchor the whole histone octamer to DNA.This explains the simultaneous existence of thenaked DNA-like DNase I cutting pattern andthe presence of histones in the remodelednucleosomes. This model is in agreement withrecent studies, showing a strong remodeling ofnucleosomes upon the action of SWI/SNF (Bazett-Jones et al., 1999; CoÃ teÂ et al., 1998; Lorch et al., 1998;Schnitzler et al., 1998; Lee et al., 1999).

In a recent report, Guyon et al. (1999) havedemonstrated a stable remodeling of taillessnucleosomes by the human SWI/SNF complex. Intheir study the authors concluded that remodelingby SWI/SNF can occur via the interactions with atailless nucleosome. This conclusion is consistentwith our ®nding that SWI/SNF does not alter theamount of histone NH2 tail-DNA contacts.

All these data suggest that SWI/SNF actsthrough the disruption of histone-structureddomain-DNA contacts, while another remodelingcomplex, NURF, seemed to require histone NH2

termini for its activity. Thus, different complexesmay use different pathways to remodel nucleo-somes (for details see Hamiche et al., 1999; Langstet al., 1999; CoÃ teÂ et al., 1998; Schnitzler et al., 1998;Lorch et al., 1998), and it will be of interest toinvestigate their mechanism of action using thelaser crosslinking approach in combination withDNase I protection analysis and EMSA exper-iments.

Histones and actively transcribed genes

Here we have shown that the binding of mul-tiple transription activators to HIV-1 nucleosomeinduces the release of signi®cant amounts of his-tone NH2 tails from nucleosomal DNA, but thatsome of the tails (about 40 %) preserve their con-tacts with DNA. In addition, we have demon-strated that SWI/SNF-induced remodeling of theHIV-1 nucleosome does not alter histone NH2 tail-DNA binding. Recently, we have also demon-strated an interaction of both hyperacetylated or


non-acetylated histone tails with DNA that persistsin the presence of ®ve simultaneously boundGAL4-AH dimers occupying the central 90 bp ofnucleosomal DNA (Mutskov et al., 1998). These, aswell as other in vitro data (Utley et al., 1997; Steger& Workman, 1997), suggest that in vivo, the pro-moter region of actively transcribed genes mightbe associated with histones in a non-canonicalnucleosomal organization, where histones areanchored to DNA mainly through their NH2 tails.Promoters organized in this way may not showthe typical microccocal nuclease digestion patternsand could exhibit enhanced accessibility to DNaseI (Steger & Workman, 1997). Indeed, the chemicalcrosslinking procedure operating through the his-tone NH2 tails used by Mirzabekov and colleagues(Nacheva et al., 1989) detected histones on both theactively transcribed non-nucleosomally organizedhsp70 gene and the non-active nucleosomally orga-nized one. Consistent with these ®ndings, UV laserCHIP experiments demonstrated the presence ofhistones on the enhancers, promoters and codingregions of actively transcribed Xenopus laevisribosomal genes (Dimitrov et al., 1990; Mutskovet al., 1996).

Materials and Methods

Preparation of DNA probes

The DNA probes containing the binding sites of theSp1, NF-kB, LEF-1 and USF transcription factors wereproduced by PCR ampli®cation of HIV-1 50LTR. Brie¯y,two sets of 30-mer primers containing either EcoRI orXbaI restriction sites at their 50 end were used for thegeneration of DNA probes with 174 bp and 164 bp DNAlength. After restriction of the PCR products by EcoRIand XbaI, the obtained 150 bp (containing nucleotidesÿ32 to ÿ181 from HIV-1 50LTR) and 160 bp (containingnucleotides ÿ25 to ÿ188 from HIV-1 LTR) DNA probeswere uniquely end-labeled, by ®lling with Klenow, andgel puri®ed. The speci®c activity of uniquely end-labeledfragments was typically 2 � 107-4 � 107 cpm/mg DNA.The probes with higher speci®c activity (typically1 � 108-1.5 � 108 cpm/mg) used for immunoprecipitationwere generated by labeling with a mixture of[a-32P]dCTP, [a-32P]dATP and [a-32P]dTTP.

Protein purification and nucleosome reconstitution

Bacterially expressed human NF-kB1 (i.e. the p50 sub-unit) and human USF were prepared according to themethod of Adams & Workman (1995). RecombinantLEF-1 was isolated from bacterial strains as described bySteger & Workman (1997). Sp1 was prepared by theexpression of full-length protein in the Bac-to-bac baculo-virus system (Life Technologies, Rockville MD). The pro-tein was puri®ed from the extract of recombinantbaculovirus-infected Sf21 cells using a wheat germ lectincolumn, essentially as described by Jackson & Tjian(1989). The preparation of SWI/SNF and chicken linkerhistone-depleted oligosomes was as described by CoÃteÂet al. (1994) and Mutskov et al. (1998), respectively. Theintegrity of the isolated proteins was controlled by poly-acrylamide gels containing SDS (Laemmli, 1970).

The reconstitution of nucleosomes by the histone octa-mer transfer method was carried out as described(Mutskov et al., 1998). A typical reconstitution reactioncontained 250 ng of labeled probe DNA and 4mg of lin-ker histone-depleted chicken erythrocyte donor nucleo-some (1:16 ratio). Under these conditions thereconstitution yield was >85 %. For EMSA and footprint-ing experiments, the ratio of probe DNA to donor coreparticles was 1:25 and the reconstitution yield wasusually closer to 95 %.

Binding reactions

Transcription factor binding

The transcription factor binding reactions were carriedout in 10 mM Hepes (pH 7.8), 50 mM KCl, 5 mM DTT,0.5 mM PMSF, 200 mg/ml bovine serum albumin (BSA),5 % (v/v) glycerol (binding buffer). A typical reaction of20 ml contained 1.5-2.5 ng (15-25 fmol) of reconstitutedend-labeled nucleosomes. Concentrated stock solutionsof transcription factors were diluted in the binding bufferimmediately prior to use and added in appropriatevolumes. The reactions were incubated at 30 �C for20 minutes. After completion of each reaction the ef®-ciency of binding was checked by EMSA.

SWI/SNF experiments

For the SWI/SNF experiments the binding buffer wasadjusted to 4 MgCl2 and 0.5 mM ATP, as required forSWI/SNF activity. SWI/SNF was added directly to thebinding reaction in the proportion of 2 ml (®nal concen-tration 10 nM) per 25 ng of nucleosomes or naked DNAand incubated at 30 �C for 45 minutes (reactions notreceiving these proteins were supplemented with theappropriate amount of SWI/SNF buffer: 40 mM Hepes(pH 7.5), 350 mM NaCl, 0.1 % (v/v) Tween, 10 mMZnCl2, 10 % glycerol, 0.5 mM DTT, anti-protease inhibi-tors (0.1 mM PMSF, 5 mg/ml aprotinin, 5 mg/ml pepsta-tin, 5 mg/ml leupeptin and 50 ng/ml insulin).

To remove SWI/SNF bound to nucleosomes, a 25-30-fold excess of core particle competitor together with 2units of apyrase was added to the reaction mixtures andthey were additionally incubated for 20 minutes at 30 �C.All binding reactions were controlled by EMSA.

EMSA and DNase I protection analysis

EMSA was performed at 4 �C in 4 % (w/v) polyacryl-amide (acrylamide to bisacrylamide, 29:1), 0.5� TBEgels. For DNase I nucleosome protection analysis thebinding reactions were supplemented with 5 mM MgCl2and digested with 20 ng of DNase I for 2 minutes atroom temperature. No additional MgCl2 was added toSWI/SNF containing samples and samples containingcompetitor unlabeled nucleosomes were treated asabove, but with 30 ng of DNase I. Reactions containingDNA only were digested with tenfold less DNase I.DNase activity was terminated by adding 80 ml of stopsolution (10 mM Tris (pH 7.6), 10 mM EDTA (pH 8.0),0.15 % SDS, 0.1 mg/ml proteinase K) and the proteinsdegraded by incubating at 37 �C for 30 minutes. TheDNA was subsequently puri®ed by phenol, precipitatedwith ethanol and loaded on 8 % polyacrylamide (acryl-amide to bisacrylamide, 19:1), 8 M urea, 1� TBE sequen-cing gels. Gels were run at 60 W constant power, dried


and exposed to a phosphorimager screen (MolecularDynamics).

UV laser irradiation

The samples were irradiated with the fourth harmo-nics (l � 266 nm, t � 5 ns) of a Surelite II Nd:YAG laser(Continuum, USA). Measurements of the pulse energywere carried out with a calibrated pyroelectrical detector(Ophir Optronics Ltd). Two types of irradiation wereused depending of the optical thickness of the solution.

Optical thin layer irradiation (optical density of theirradiated sample at 260 nm (E260)<0.1)

The transcription factor-nucleosome complexes usedfor immunoprecipitation (Figure 3) were irradiatedunder these conditions since their absorbancy at 260 nmwas <0.1. DNA-protein crosslinking was performed byirradiation of 20 ml binding reaction aliquots with onepulse of 0.25 J/cm2 (eight absorbed photons per nucleo-base) in a 0.65 ml siliconized Eppendorf tube (the size ofthe laser beam was adjusted to ®t perfectly the surfacearea of the sample (Mutskov et al., 1998)). Under theseconditions about 30 % of the DNA was crosslinked tohistones as determined by phenol assay (Mutskov et al.,1998).

Optical thick layer irradiation (E260>1)

The irradiation of the binding reactions containingSWI/SNF (Figure 6) was performed under optical thicklayer approximation, since the presence of 0.5 mM ATPincreases the optical density of the samples to E266 � 6.5.To the control reactions, dCTP, instead of ATP, wasadded to the same optical density. The reaction mixture(200 ml) was irradiated in a rectangular silica cell underconstant stirring with 2 J total energy, which corre-sponds to 21 absorbed photons per nucleobase (it istaken into account that the extinction of the bases inDNA at 266 nm, E266 � 6000 Mÿ1 cmÿ1, is on averageabout 2.1 times smaller than that of ATP,E266 � 12800 Mÿ1 cmÿ1) with a pulse energy density of0.15 J cmÿ2. It should be noted that the total number ofabsorbed photons in the case of the SWI/SNF bindingreactions (21 absorbed photons) is 2.6 times higher thanthat of thin optical samples (eight absorbed photons),since, due to the biphotonic character of the photoreac-tion, the ef®ciency of crosslinking upon irradiation of anoptical thick layer is lower than that of an optical thinlayer (Nikogosyan & Lethokov, 1983; Nikogosyan, 1990).This energy of irradiation induces the crosslinking of30 % of nucleosomal DNA to histones as determined byphenol assay, i.e. the same histone-DNA crosslinkingyield as for a thin layer optical sample.

After each irradiation experiment the integrity of thesamples was checked by EMSA. No changes in the elec-trophoretic mobility of the different nucleoprotein com-plexes was observed, suggesting that irradiation-induceddamage did not alter their overall structure.

Preparation and immunopurification of antibodies

Antisera against core histones H2A, H2B and H4 wereobtained by injecting rabbits at multiple sites with200 mg of individual histone-RNA complexes in completeFreund's adjuvant (Angelov et al., 1988). Two weekslater the procedure was repeated with the same dose,

but with incomplete adjuvant. After a period of 10-14days an intravenous injection with the same quantity ofantigen was administrated. The rabbits were bled, thesera were taken and immunopuri®ed antibodies wereprepared by af®nity chromatography with individualhistones conjugated to CNBr-Sepharose (Russanova et al.,1987). The speci®city of the antibodies was controlled byimmunoblotting.

Immunoblotting

Histones were separated on 18 % polyacrylamide gelscontaining SDS (Laemmli, 1970), and gels were trans-ferred to nitrocellulose ®lters. Immunodetection was per-formed essentially as described (Dimitrov & Wolffe,1997).

Immunoprecipitation procedure (laser CHIP)

The individual covalent histone-DNA complexes wereimmunoprecipitated as described by Mutskov et al.(1998), but with minor modi®cations. Aliquots (30 ml) ofprotein-A Sepharose (Pharmacia) were resuspended in0.8 ml of a solution of 300 mg/ml total histone in 50 mMTris-HCl (pH 8.0), 2 M NaCl, and shaken for one hour atroom temperature to block sites of non-speci®c absorp-tion. The resin was pelleted for 30 seconds in a bench-top centrifuge and washed twice in 1� antibody buffer(50 mM Hepes (pH 7.5), 2 M NaCl, 0.1 % SDS, 1 % TritonX-100, 1 % (w/v) deoxycholate, 5 mM EDTA (pH 8.0)).The pelleted resin (30 seconds in a bench-top centrifuge)was resuspended in 0.8 ml of 1� antibody buffer sol-ution containing 50 ml of irradiated binding reactions(consisting of 4-5 ng of reconstituted labeled nucleo-somes (5 � 105-6 � 105 cpm), 50-60 ng of carrier H1-depleted oligosomes and the transcription factors orSWI/SNF complex) and the speci®c antibody at a ratioof 1:2.5 of antibody to the labeled and carrier particles.The suspension was incubated with gentle shaking forthree hours at room temperature, washed ®ve times with1� antibody buffer and three times with 50 mM Hepes(pH 7.5), 0.15 M NaCl, 5 mM EDTA. The amount of thespeci®cally immunoprecipitated covalent histone-DNAcomplexes was determined by Cerenkov counting.

In the case of additivity of the action of each nucleo-some-bound transcription factor on the histone NH2 tailrelease, the total effect �calculated was calculated as�calculated � (ESp1 � ENF-kB � ELEF-1 � EUSF)/4 where Esp1,

ENF-kB, ELEF-1 and EUSF were the values of the experimen-tally determined effects (Figure 3) of Sp1, NF-kB, LEF-1and USF, respectively, when bound individually to thenucleosome.

Acknowledgments

This work was mainly supported by Sidaction (con-tract 991249/23026-01-00/AO10-1). We thank Drs J.Workman and D. Steger for the expression vectors forNF-kB, USF, and LEF-1 proteins as well as for advice fortheir puri®cation, E. Verdin for a plasmid construct con-taining HIV-1 50LTR, F. Hans and M. Callanan for criticalcomments on the manuscript and S. Allard for technicalassistance. We also appreciate the support of Dr Jean-Jacques Lawrence throughout the course of this work.J. C. acknowledges a Canadian MRC scholarship andoperating grant.


References

Adams, C. C. & Workman, J. L. (1995). The binding ofdisparate transcription factors to nucleosomal DNAis inherently cooperative. Mol. Cell. Biol. 15, 1405-1421.

Angelov, D., Stefanovsky, V. Y., Dimitrov, S. I.,Russanova, V. R., Keskinova, E. & Pashev, I. G.(1988). Protein-DNA crosslinking in reconstitutednucleohistone, nuclei and whole cells by picosecondUV laser irradiation. Nucl. Acids Res. 16, 4525-4538.

Arents, G., Burlingame, R. W., Wang, B.-C., Love, W. E.& Moudrianakis, E. N. (1991). The nucleosomal corehistone octamer at 3.1 AÊ resolution: a tripartite pro-tein assembly and a left-handed superhelix. Proc.Natl Acad. Sci. USA, 88, 10148-10152.

Bazett-Jones, D. P., CoÃ teÂ, J., Lander, C. C., Peterson, C. L.& Workman, J. L. (1999). The SWI/SNF complexcreates loop domains in DNA and polynucleosomearrays and can disrupt DNA-histone contactswithin these domains. Mol. Cell. Biol. 19, 1470-1478.

Cary, P. D., Moss, T. & Bradbury, M. (1978). High-resol-ution-proton-magnetic-resonance studies of chroma-tin core particle. Eur. J. Biochem. 89, 475-482.

CoÃ teÂ, J., Quinn, J., Workman, J. & Peterson, C. L. (1994).Stimulation of GAL 4 derivative binding to nucleo-somal DNA by the yeast SWI/SNF complex.Science, 265, 53-60.

CoÃ teÂ, J., Peterson, C. L. & Workman, J. L. (1998). Pertur-bation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhan-cing subsequent transcription factor binding. Proc.Natl Acad. Sci. USA, 95, 4947-4952.

Dimitrov, S. I. & Wolffe, A. P. (1997). Fine resolution ofhistones by two-dimensional polyacrylamide gelelectrophoresis: developmental implications.METHODS: A Companion to Methods Enzymol. 12,57-61.

Dimitrov, S. I., Stefanovsky, V., Karagyozov, L.,Angelov, D. A. & Pashev, I. G. (1990). The enhan-cers and promoters of Xenopus laevis ribosomalspacer are associated with histones upon activetranscription of the ribosomal genes. Nucl. AcidsRes. 18, 6393-6397.

Dimitrov, S. I., Tateossyan, H. N., Stefanovsky, V. Y.,Russanova, V. R., Karagyozov, L. & Pashev, I. G.(1992). Binding of histones to Xenopus laevis riboso-mal genes with different level of expression. Eur. J.Biochem. 204, 977-981.

Edmonson, D. G., Smith, M. M. & Roth, S. I. (1996).Repression domain of yeast global repressor Tup1interacts directly with histones H3 and H4. GenesDev. 10, 1247-1259.

Georgel, P. T., Tsukiyama, T. & Wu, K. (1997). Role ofhistone tails in nucleosome remodeling by Droso-phila NURF. EMBO J. 16, 4717-4726.

Guyon, J. R., Narlikar, G. J., Sif, S. & Kingston, R. E.(1999). Stable remodeling of tailless nucleosomes bythe human SWI-SNF complex. Mol. Cell. Biol. 19,2088-2097.

Hamiche, A., Sandaltzopoulos, R., Gdula, D. A. & Wu,C. (1999). ATP-dependent histone octamer slidingmediated by the chromatin remodeling complexNURF. Cell, 97, 833-842.

Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S. M.& Grunstein, M. (1995). Histone H3 and H4 terminiinteract with SIR3 and SIR4 proteins: a molecularmodel for the formation of heterochromatin inyeast. Cell, 80, 583-592.

Hockensmith, J. W., Kubasek, W. L., Vorachek, W. R. &von Hippel, P. H. (1986). Laser cross-linking ofnucleic acids to proteins. Methodology and ®rstapplication to the phage T4 DNA replication sys-tem. J. Biol. Chem. 261, 3512-3518.

Hockensmith, J. W., Kubashek, W. L., Vorachek, W. R.,Evertz, E. M. & von Hippel, P. H. (1991). Lasercross-linking of protein-nucleic complexes. MethodsEnzymol. 208, 211-236.

Hockensmith, J. W., Kubasek, W. L., Evertz, E. M.,Mesner, L. D. & von Hippel, P. H. (1993a). Lasercrosslinking of proteins to nucleic acids. II. Inter-actions of the bacteriophage T4 DNA replicationpolymerase accessory proteins complex with DNA.J. Biol. Chem. 268, 15721-15730.

Hockensmith, J. W., Kubasek, W. L., Vorachek, W. R. &von Hippel, P. H. (1993b). Laser cross-linking ofproteins to nucleic acids. I. Examining physical par-ameters of protein-nucleic acids complexes. J. Biol.Chem. 268, 15712-15720.

Imbalzano, A. N., Schnitzler, G. R. & Kingston, R. E.(1996). Nucleosome disruption by human SWI/SNFis maintained in the absence of continued ATPhydrolysis. J. Biol. Chem. 271, 20726-20733.

Jackson, S. P. & Tjian, R. (1989). Puri®cation and anal-ysis of RNA polymerase II transcription factors byusing wheat germ agglutinin af®nity chromatog-raphy. Proc. Natl Acad. Sci. USA, 86, 1781-1785.

Kornberg, R. D. & Lorch, Y. (1999). Chromatin-modify-ing and -remodeling complexes. Curr. Opin. Genet.Dev. 9, 148-151.

Laemmli, U. K. (1970). Cleavage of structural proteinsduring the assembly of the head of bacteriophageT4. Nature, 227, 680-685.

Langst, G., Bonte, E. J., Corona, D. F. V. & Becker, P. B.(1999). Nucleosome movement by CHRAC andISWI without disruption or trans-displacement ofthe histone octamer. Cell, 97, 843-852.

Lee, D. Y., Hayes, J. J., Pruss, D. & Wolffe, A. P. (1993).A positive role for histone acetylation in transcrip-tion factor access to nucleosomal DNA. Cell, 72, 73-84.

Lee, K.-M., Sif, S., Kingston, R. E. & Hayes, J. S. (1999).hSWI/SNF disrupts interactions between the H2AN-terminal tail and nucleosomal DNA. Biochemistry,38, 8423-8429.

Lorch, Y., Cairns, B. R., Zhang, M. & Kornberg, R. D.(1998). Activated RSC-nucleosome complex andpersistently altered form of the nucleosome. Cell,94, 29-34.

Luger, K. & Richmond, T. J. (1998). The histone tails ofthe nucleosome. Curr. Opin. Genet. Dev. 8, 140-146.

Luger, K., MaÈder, A. W., Richmond, R. K., Sargent, D. F.& Richmond, T. J. (1997). Crystal structure of thenucleosome core particle at 2.8 AÊ resolution. Nature,389, 251-260.

Moss, T., Dimitrov, S. I. & Houde, D. (1997). UV cross-linking of proteins to DNA. METHODS: A Compani-nion to Methods Enzymol. 11, 225-234.

Mutskov, V. J., Russanova, V., Dimitrov, S. I. & Pashev,I. G. (1996). Histones associated with non-nucleoso-mal rat-ribosomal genes are acetylated, while thosebound to nucleosome organized gene copies arenot. J. Biol. Chem. 271, 11852-11857.

Mutskov, V., Gerber, D., Angelov, D., Ausio, J.,Workman, J. & Dimitrov, S. (1998). Persistent inter-actions of core histone tails with nucleosomal DNAfollowing acetylation and transcription factor bind-ing. Mol. Cell. Biol. 18, 6293-6304.


Nacheva, G. A., Gushin, D. Y., Preobrazhenskaya, O. V.,Karpov, V. I., Ebralidze, K. K. & Mirzabekov, A. D.(1989). Changes in the pattern of histone binding toDNA upon transcription activation. Cell, 58, 27-36.

Nikogosyan, D. N. (1990). Two-quantum UV photo-chemistry of nucleic acids: comparison with conven-tional low-intensity UV photochemistry andradiation chemistry. Int. J. Radiat. Biol. 57, 233-299.

Nikogosyan, D. & Lethokov, V. S. (1983). Nonlinearlaser photophysics, photochemistry and photobiol-ogy of nucleic acids. Riv. Nuovo Cimento, 3, 1-74.

Pashev, I. G., Dimitrov, S. I. & Angelov, D. (1991).Laser-induced protein-DNA crosslinking. TrendsBiochem. Sci. 16, 323-326.

Pazin, M. G. & Kadonaga, J. T. (1997). SWI2/SNF2 andrelated proteins: ATP-driven motors that disruptprotein-DNA interactions? Cell, 88, 737-740.

Peterson, C. L. & Tamkun, J. W. (1995). The SWI-SNFcomplex: a chromatin remodeling machine? Trends.Biochem. Sci. 20, 145-148.

Pollard, K. J. & Peterson, C. L. (1998). Chromatin remo-deling: a marriage between two families. BioEssays,20, 771-780.

Roth, S. Y. & Allis, C. D. (1996). Histone acetylation andchromatin assembly: a single escort, multipledances? Cell, 87, 5-8.

Russanova, V. R., Dimitrov, S. I., Makarov, V. L. &Pashev, I. G. (1987). Accessibility of the globulardomain of histones H1 and H5 to antibodies uponfolding of chromatin. Eur. J. Biochem. 167, 321-326.

Schnitzler, G., Sif, S. & Kingston, R. E. (1998). HumanSWI/SNF interconverts a nucleosome between itsbasic state and a stable remodeled state. Cell, 94, 17-27.

Sheridan, P. L., Mayall, T. P., Verdin, E. & Jones, K. A.(1997). Histone acetyltransferases regulate HIV-1enhancer activity in vitro. Genes Dev. 11, 3327-3340.

Stefanovsky, V., Dimitrov, S. I., Russanova, V. R.,Angelov, D. & Pashev, I. G. (1989). Laser-inducedcrosslinking of histones to DNA in chromatin andcore particles: implications in studying histone-DNA interactions. Nucl. Acids Res. 23, 10069-10081.

Steger, D. J. & Workman, J. L. (1997). Stable co-occu-pancy of transcription factors and histones at theHIV-1 enhancer. EMBO J. 16, 2463-2472.

Steger, D. J., Eberharter, A., John, S., Grant, P. A. &Workman, J. L. (1998). Puri®ed histone acetyltrans-ferase complexes stimulate HIV-1 transcription frompreassembled nucleosomal arrays. Proc. Natl Acad.Sci. USA, 95, 12924-12929.

Sudarsanam, P., Cao, Y., Wu, L., Laurent, B. C. &Winston, F. (1999). The nucleosome remodelingcomplex, Snf/Swi, is required for the maintenanceof transcription in vivo and is partially redundantwith the histone acetyltransferase, Gcn5. EMBO J.18, 3101-3106.

Travers, A. (1999). An engine for nucleosome remodel-ing. Cell, 96, 311-314.

Utley, R. T., CoÃ teÂ, J., Owen-Hughes, T. & Workman, J. L.(1997). SWI/SNF stimulates the formation of dispa-rate activator-nucleosome complexes but is partiallyredundant with cooperative binding. J. Biol. Chem.272, 12642-12649.

van Holde, K. E. (1988). Chromatin, Springer-Verlag,Berlin.

van Lint, C., Emiliani, S., Ott, M. & Verdin, E. (1996).Transcriptional activation and chromatin remodel-ing of the HIV-1 promoter in response to histoneacetylation. EMBO J. 15, 1112-1120.

Verdin, E. (1991). DNase I-hypersensitive sites areassociated with both long terminal repeats and withthe intragenic enhancer of integrated human immu-node®ciency virus type 1. J. Virol. 65, 6790-6799.

Verdin, E., Paras, P. J. & van Lint, C. (1993). Chromatindisruption in the promoter of human immunode®-ciency virus type 1 during transcriptional activation.EMBO J. 12, 3249-3259.

Vettese-Dadey, M., Grant, P. A., Hebbes, T. R., Crane-Robinson, C., Allis, C. D. & Workman, J. L. (1996).Acetylation of histone H4 plays a primary role inenhancing transcription factor binding to nucleoso-mal DNA in vitro. EMBO J. 15, 2508-2518.

Wade, P. A., Pruss, D. & Wolffe, A. P. (1997). Histoneacetylation: chromatin in action. Trends Biochem. Sci.22, 128-132.

Widlak, P., Gaynor, R. B. & Garrard, W. T. (1997).In vitro chromatin assembly of the HIV-1 promoter.J. Biol. Chem. 272, 17654-17661.

Wilson, C. J., Chao, D. M., Imbalzano, A. M., Schnitzler,G. R., Kingston, R. E. & Young, R. A. (1996). RNApolymerase II holoenzyme contains SWI/SNF regu-lators involved in chromatin remodeling. Cell, 84,235-244.

Winston, F. & Carlson, M. (1992). Yeast SNF/SWI tran-scriptional activators and the SPT/SIN chromatinconnection. Trends Genet. 8, 387-391.

Wolffe, A. P. & Pruss, D. (1996). Targeted chromatindisruption: transcription regulators that acetylatehistones. Cell, 84, 817-819.

Edited by M. Yaniv

(Received 17 February 2000; received in revised form 23 June 2000; accepted 21 July 2000)

Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex...

Documents

Transcript of Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex...