Analysis of Transcription Factor-Mediated Remodeling of Nucleosomal Arrays in a Purified System

METHODS: A Companion to Methods in Enzymology 12, 276–285 (1997)Article No. ME970479

Analysis of Transcription Factor-MediatedRemodeling of Nucleosomal Arraysin a Purified System

David J. Steger, Tom Owen-Hughes, Sam John, and Jerry L. Workman1

Department of Biochemistry and Molecular Biology, The Center for Gene Regulation,The Pennsylvania State University, University Park, Pennsylvania 16802-4500

ture, suggesting that the primary steps leading toAn early step in a pathway leading to transcriptional initia- transcriptional initiation involve interactions be-

tion involves the rearrangement of chromatin at gene regula- tween activators and nucleosomes (3).tory sequences. To study this process, we have developed a To counteract nucleosome-mediated repression,biochemical system analyzing the interactions between chro- transcription factors must compete with histones formatin templates composed of arrays of positioned nucleo- sites on the DNA. Many of the mechanisms govern-somes and sequence-specific transcriptional activators. ing this process have been established through bio-Here, a procedure is presented for the assembly of nucleoso- chemical studies utilizing nucleosome core particlesmal arrays on DNA fragments containing synthetic and natural (for review, see 4). For example, the affinity of tran-gene sequences inserted within tandem repeats of sea urchin scription factors for nucleosomal DNA is determined5S rDNA. We also provide methods for the use of these tem- by the location of binding sites within nucleosomesplates in transcription factor-binding assays, as well as exper- and by the cooperative interactions of multiple pro-imental data illustrating the efficacy of such analyses to un- teins recognizing sequences contained within thecover mechanisms directing factor-mediated nucleosome same nucleosomes. Furthermore, distinct types ofremodeling. q 1997 Academic Press protein complexes facilitate transcription factor

binding to nucleosomal DNA. Chromatin remodelingactivities such as SWI/SNF, NURF, and RSC har-ness the energy of ATP to disrupt histone–DNA in-teractions (5, 6). By acetylating lysine residues on

Nucleosomes negatively regulate gene expression the amino-terminal tails of the core histone proteins,by restricting access of the transcriptional machin- histone acetyltransferases also alter histone–DNAery to the DNA (reviewed in 1). Thus, it is not sur- interactions to enhance factor access to sites inprising that the chromatin structure at enhancer nucleosomes (7).and promoter regions is often reconfigured prior to, The simplicity of biochemical studies using nucleo-or concurrently with, the induction of gene transcrip- some core particles allows for a direct determinationtion (reviewed in 2). Importantly, the sequence-spe- of the effects of transcription factor binding oncific binding of transcriptional activators has been nucleosomal structure. However, the binding ofdirectly linked to the remodeling of chromatin struc- transcriptional activators within a cellular context

occurs on DNA incorporated into long arrays ofnucleosomes. As a result, changes in the native chro-1 To whom correspondence should be addressed. Fax: (814) 863-

0099. E-mail: [email protected]. matin structure of gene regulatory regions are gen-

276 1046-2023/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

AID Methods A 0479 / 6714$$$141 06-11-97 23:26:33 metaas AP: Methods A

277TRANSCRIPTION FACTOR BINDING TO NUCLEOSOMAL ARRAYS

erally detected as sites with increased sensitivities sequences we have placed between 5S rDNA repeats.The 5xGAL4/5S nucleosomal array has served as ato nucleases relative to the neighboring chromatin.

A biochemical analysis of the mechanisms directing useful model system to elucidate the mechanismsemployed by transcription factors in reconfiguringchromatin remodeling, therefore, requires that tem-

plates harboring nucleosomal arrays be employed. chromatin in the process of gaining access to recogni-tion sequences (13). The HIV/5S array was con-The assembly of nucleosomes onto defined DNA

templates in vitro is currently achieved by two dis- structed to biochemically recreate aspects of the na-tive chromatin organization for the HIV-1 5* LTR.tinct approaches. One approach utilizes cell-free pro-

tein extracts from Drosophila embryos (8) or Xeno- An analysis of transcription factor binding to thistemplate has identified key roles played by specificpus oocytes (9) for nucleosome reconstitution. These

extracts contain enzymatic activities that efficiently proteins in reconfiguring the nucleosomal structurein a manner resembling the in vivo architecture (14).assemble evenly spaced arrays of nucleosomes onto

plasmid DNA. However, a heterogeneous populationof templates often exists within a single reconstitu-tion reaction, since nucleosomal positions can vary

MATERIALS AND METHODSon the same DNA from molecule to molecule. More-over, chromatin studies are generally carried out inthe presence of the reconstitution extract, which con- In this section, we describe the preparation of

polynucleosome-reconstituted arrays and their usetains a large assortment of undefined proteins.Thus, in some instances, the use of impure reconsti- in binding reactions with transcription factors in

vitro. Assembly of nucleosomes onto the 5S arraytution systems may ultimately limit the extent ofinterpretation of the experimental results. DNAs schematically illustrated in Fig. 1 is achieved

by histone octamer transfer (10). In this method, aA complementary approach, which is the topic ofdiscussion here, uses a highly purified system to as- radiolabeled 5S array fragment is combined with a

large molar excess of purified HeLa oligonucleosomesemble arrays of positioned nucleosomes onto DNAfragments. Nucleosome reconstitution is performed cores, i.e., H1-depleted nucleosomal DNA of approxi-

mately three to five nucleosomes. The mixture isby histone octamer transfer, which relies on changesin salt concentration to assemble nucleosomes onto placed in high salt so that histones dissociate from

the HeLa DNA. As the salt concentration is slowlyexperimental templates (10). In general, this tech-nique does not yield evenly spaced or positioned decreased, histones reassociate with DNA to form

nucleosomes, and, because the probe DNA repre-nucleosomes on DNA fragments large enough to ac-commodate multiple nucleosomes. However, an ob- sents a small proportion of the total DNA, the major-

ity of the probe (typicallyú90%) is incorporated intoservation made many years ago by Simpson andStafford (11) revealed that a DNA segment of a 5S nucleosomes. In addition to being a very simple pro-

cedure, histone octamer transfer efficiently assem-rRNA gene from the sea urchin Lytechinus variega-tus strongly positions a histone octamer when recon- bles nucleosomes onto the probe DNA, which allows

for direct use of the reconstitutes in binding assaysstituted with histones in vitro using purified compo-nents. Moreover, Simpson et al. (12) went on to without further purification.

We do not present a protocol for the preparationdemonstrate that reconstitution of direct repeats of5S rDNA yields a continuous array of positioned of purified HeLa oligonucleosome cores, as this has

been described in detail elsewhere (15). Further-nucleosomes, the spacing of which could be variedby adjusting the 5S rDNA repeat length. We have more, we do not discuss the purification of transcrip-

tion factors, as those used in the experiments de-modified this system to produce nucleosomal arraysbearing transcription factor-binding sites by placing scribed below may not apply to the interests of the

reader. It is important to note, however, that thetandem 5S rDNA repeats on both sides of targetDNA sequences of interest. Upon reconstitution, the isolation of large amounts of factors is usually re-

quired since binding to nucleosomes generally occurs5S rDNA repeats adopt positioned nucleosomes thatsubsequently encourage the positioning of a nucleo- with a reduced affinity relative to that of DNA.

Protocols for radiolabeling DNA can be found insome on the central sequence in phase with theneighboring nucleosomes. Figure 1 shows a sche- many molecular biology manuals. We end-label a 5S

array by enzymatically digesting plasmid DNA to pro-matic of the synthetic and natural regulatory gene


278 STEGER ET AL.

duce a 5* overhang at a single site directly flanking 13 ml of 50 mM Hepes, pH 7.5, 1 mM EDTA, 5 mM

DTT, and 0.5 mM PMSF, with 30-min incubationsone end of the array, and we subsequently fill in theoverhang with Klenow in the presence of [a-32P]dATP. at 307C for each dilution step. The reaction is then

brought to 0.1 M NaCl by adding 67 ml of 10 mMDigestion of the labeled plasmid DNA with a secondrestriction enzyme, which cuts at a site flanking the Tris–HCl, pH 7.5, 1 mM EDTA, 0.1% NP-40, 0.5

mM PMSF, 20% glycerol, and 100 mg/ml BSA andopposite end of the array from that of the first enzyme,releases the 5S array fragment from background vec- incubating for 30 min at 307C. The nucleosome-re-

constituted array remains stable for at least 4 weekstor sequence and enables gel purification by electro-phoresis through low-melting-point agarose and isola- when stored at 47C. For a mock reconstitution, the

probe DNA is added to a transfer reaction after thetion by b-agarase I treatment.Nucleosomes are assembled onto 5S array frag- salt has been decreased to 0.1 M NaCl. Therefore,

all of the components in a mock-reconstituted sam-ments by histone octamer transfer as described byCote et al. (15). In short, Ç40 pmol of HeLa nucleo- ple are the same as that for a normal reconstitution

reaction; however, octamer transfer does not occur,somes (corresponding to 5 mg of DNA from the HeLanucleosomes) are mixed at 377C for 20 min withÇ0.2 so the probe DNA remains free of histones.

Purified transcription factors and chromatin re-pmol of the radiolabeled 5S array DNA (correspond-ing to Ç500,000 cpm by Cerenkov counting) in a modeling activities are added to the nucleosome-re-

constituted arrays to determine whether potentialfinal volume of 10 ml containing 50 mM Hepes, pH7.5, 1 M NaCl, 1 mM EDTA, 5 mM DTT, and 0.5 mM interactions reconfigure nucleosomal structure. Typ-

ically, 0.5 ml of the octamer transfer reaction (i.e.,PMSF. Because each of the 5xGAL4/5S and HIV/5Sfragments harbors 11 nucleosomes when fully recon- 2500 cpm) is mixed with factors, so that a binding

reaction containsÇ1 fmol of reconstituted probe andstituted, the ratio of donor nucleosomes to octamer-binding sites on the probe is nearly 20:1. It is benefi- a total of Ç200 fmol of nucleosomes. Binding reac-

tions are performed at 307C for 30 min, in a volumecial to keep this ratio high, since efficient reconstitu-tion occurs at molar ratios greater than 10:1 of donor of 20 ml containing 10 mM Hepes, pH 7.8, 50 mM

NaCl (or KCl), 5 mM DTT, 0.5 mM PMSF, 0.25 mg/nucleosomes to probe nucleosomes. The transfer re-action is serially diluted by adding 1.8, 3.5, 4.7, and ml BSA, and 5% glycerol.

FIG. 1. Schematic of the DNA fragments used to assemble arrays of positioned nucleosomes. A 5-GAL4-site fragment or HIV-1 5*

LTR sequence is flanked on both sides by five direct repeats of a 208-bp 5S rDNA fragment to produce the 5xGAL4/5S and HIV/5Sarray DNAs, respectively. The 5-GAL4-site and HIV-1 fragments are both approximately 208 bp to maintain the repeat lengthestablished by the 5S sequences. The GAL4-binding sites are centered within the 5-GAL4-site DNA, while the HIV-1 sequencecontains binding sites for Sp1, NF-kB, LEF-1, ETS-1, and USF. Three SalI restriction enzyme sites are present at both ends of the5-GAL4-site fragment, which allows this DNA to be excised from the 5S repeats for analysis in the nucleosome cut-out assay (discussedbelow). The XhoI sites flanking the HIV-1 sequence serve the same role in the HIV/5S DNA. The EcoRI sites present at the junctionsbetween 5S repeats and the asymmetric positioning of a nucleosome on the 5S DNA are indicated.



Binding reactions can be assayed by several tech- displacing histones. As opposed to discrete, sub-nucleosomal hypersensitive sites resulting from theniques. For example, interactions between tran-

scriptional activators and the nucleosomal array can disruption of histone–DNA contacts, remodeling ofthis nature should, in principle, greatly enhancebe directly monitored by electrophoretic mobility

shift analysis (EMSA). Samples are electrophoresed DNase I cutting throughout the entire nucleosome-length sequence. In practice, however, the removalthrough Ç0.7% agarose gels, and the gels are fixed

(by soaking in a solution composed of 10% acetic acid of histones from factor-bound sequences does notnecessarily cause the entire nucleosomal domain toand 10% methanol), dried, and exposed to film. A

slower migration rate for the array in reactions con- become DNase I hypersensitive, since the boundtranscription factors substitute for histones in pro-taining activators versus that for a sample with only

the polynucleosomal template indicates binding. tecting the DNA from nuclease attack. Thus, to ob-tain an indication of histone displacement based onAlthough it is simple, EMSA provides no informa-

tion regarding the specificity of factor binding or DNase I analysis, removal of the bound transcriptionfactors from the 5S array is required prior towhether such binding alters nucleosomal structure.

To address these issues, binding reactions are sub- nuclease treatment. To achieve this, factors are com-peted off the nucleosome arrays by oligonucleotidejected to limited MNase or DNase I digestion,

and the digestion products are resolved by agarose challenge. Following the binding reaction incubationperiod, 1 ml of an oligonucleotide competition mix isgel electrophoresis. These nucleases preferentially

cleave 5S nucleosomal arrays in the linker DNA be- added containing 2 M NaCl and Ç10-fold molar ex-cess relative to factors of double-stranded oligonucle-tween nucleosomes, resulting in an alternating pat-

tern of cutting and protection. Nucleosome remodel- otides representing the binding sites for the factorsincluded in the experiment. Importantly, the salting due to the binding of transcription factors is

detected as an increase in nuclease sensitivity in conditions used for this procedure do not promotethe transfer of displaced histones back onto theregions occupied by histones. In our experience, re-

modeled nucleosomes are more susceptible to DNase probe DNA after the removal of factors. The competi-tion step is performed at 377C for 1 h. Increasing theI activity than MNase, which may result from the

ability of DNase I to more efficiently cleave DNA salt concentration and temperature of the bindingreactions increases the rate at which factors dissoci-sequestered within nucleosomes than MNase. Fur-

thermore, sequence-specific binding is indicated by ate from the DNA, and the large molar excess ofoligonucleotides immediately traps dissociated fac-an unaltered digestion profile for the 5S nucleo-

somes. The DNase I hypersensitive sites are, there- tors before they can rebind to the probe DNA. Someprotein–DNA complexes are extremely stable sofore, localized to the unique nucleosome harboring

recognition sequences for the transcription factors that additional measures must be taken for theirdisruption. For example, to compete GAL4-AH off(e.g., the 5xGAL4 site or the HIV nucleosome). Al-

though the exact conditions may vary from experi- the DNA, 50 mM MgCl2 is also present in the compe-tition mix to give a final concentration of 2.5 mM.ment to experiment, we obtained partial nuclease

digestion of the nucleosome-reconstituted arrays by Upon completion of the oligonucleotide competitionstep, samples are subjected to DNase I treatment.treating binding reactions for 1 min at RT with 2 ml

of a solution consisting of either 0.02 mU/ml MNase Because the competitor oligonucleotides are presentat high concentrations, the DNase I digestion condi-(Sigma), 30 mM CaCl2 or 0.125 U/ml DNase I (Boeh-

ringer Mannheim), 50 mM MgCl2. Nuclease activity tions must be increased Ç10-fold relative to reac-tions that do not undergo the competition step.is terminated by adding 20 ml stop mix (20 mM Tris–

HCl, pH 7.5, 50 mM EDTA, 1% SDS, 0.25 mg/ml Moreover, to keep the oligonucleotides from actingas histone sinks, their lengths are restricted to 20–tRNA, 0.2 mg/ml proteinase K), and the proteins are

degraded by incubating at 507C for at least 1 h. Fol- 30 bp.The production of a nucleosome-length DHSlowing ethanol precipitation, the DNA is electropho-

resed through 1–2% agarose gels, and the gels are within the 5S array suggests the absence of histones.However, to directly determine whether histones areprocessed as described above for the EMSA.

It is often of interest to investigate whether the present at a DHS, an assay not dependent onnuclease sensitivity is required. As a result, theconcerted actions of transcription factors and nucleo-

some-remodeling activities reconfigure the array by nucleosome cut-out assay was devised. In this tech-


280 STEGER ET AL.

nique binding reactions that have passed through and that the HIV nucleosome is in phase with theneighboring nucleosomes.the oligonucleotide competition step are treated with

a restriction endonuclease that releases the central DNase I treatment of the nucleosome-reconstitu-ted HIV/5S array produces a digestion patternDNA fragment harboring the factor-binding sites

(see Fig. 1 for details). The DNA is then subjected nearly identical to that of MNase (Fig. 2, comparelanes 4–6 with lanes 8–10). DNase I digestion ofto EMSA to distinguish free DNA from nucleosome

cores, which directly reveals the absence or the pres- naked HIV/5S DNA yields a smear of bands (Fig.2, lanes 11–13), confirming that the distinct ladderence of histones on the excised array fragment.

EMSA is performed in 4% polyacrylamide (29:1 of cut sites observed for the reconstituted array re-sults from the association of histones with DNA toacrylamide:bisacrylamide), 0.51 TBE gels. Unlike

the protocols employing DNase I or MNase, the form nucleosomes. Moreover, a comparison of theconditions used to digest the histone-free andnucleosome cut-out assay requires that the probe

DNA be internally radiolabeled at the central frag- nucleosomal DNAs provides a measure for the levelof nucleosome reconstitution. Under similar diges-ment excised by restriction enzyme digestion. This is

achieved by treating plasmid DNA with a restriction tion conditions (i.e., lanes 13, 8), DNase I reducesenzyme that cuts uniquely within the central frag-ment, dephosphorylating the DNA, rephosphorylat-ing in the presence of [g-32P]ATP, ligating the endstogether, and then isolating the array fragment fromthe vector sequence as described above for end-label-ing (for example, see 13).

RESULTS AND DISCUSSION

Determination of Nucleosomal Array Structure

To begin an investigation of transcription factorbinding and remodeling of nucleosome-reconstituted5S templates, it is important to determine thatnucleosomes are actually present at each 5S DNArepeat and the central DNA fragment bearing factor-binding sites. The nucleosomal architecture for DNAis indicated by MNase, which cleaves DNA in thelinker regions between nucleosomes much more effi-ciently than within nucleosomes, to produce an al-ternating pattern of cutting and protection. An ex-ample of this type of analysis is shown in Fig. 2, inwhich MNase digestion of the nucleosome-reconsti-tuted HIV/5S array generates 10 discrete cut sitesspaced evenly apart by regions inaccessible toMNase activity (Fig. 2, lanes 4–6). MNase treatmentof histone-free DNA reveals that a smaller amountof enzyme is able to readily cleave throughout the FIG. 2. Nucleosome assembly of the HIV/5S DNA template pro-entire DNA fragment (Fig. 2, lanes 1–3). Thus, the duces an array of positioned nucleosomes. Mock-reconstituted

(DNA) and nucleosome-assembled (nucl.) HIV/5S DNAs wereMNase digestion profile for the nucleosome-assem-treated with either MNase or DNase I, and the digestion productsbled array is consistent with 11 uniformly positionedwere resolved by agarose gel electrophoresis. The amount ofnucleosomes occupying the DNA. Moreover, compar-MNase or DNase I added to each lane is indicated. For eachison of the MNase profile with the EcoRI digestion quantity of MNase or DNase I assayed, samples were incubated

ladder for the HIV/5S DNA (Fig. 2, lane 7) indicates for 0.33, 1, and 3 min. EcoRI digestion under limiting conditionsof the HIV/5S DNA fragment is shown in lane 7.that the 5S nucleosomes are positioned as expected



the majority of the naked DNA to small fragments GAL4-AH is confirmed, since addition of this proteinprotects the DNA from nuclease attack only in theof several hundred basepairs in length (Fig. 2, lane

13), whereas the extent of its cutting within the region bearing the 5-GAL4-binding sites (Fig. 3B,lane 2). When GAL4-AH is incubated with thenucleosomal array is much less, such that most of

the DNA remains of the parental size (Fig. 2, lane nucleosome-reconstituted array, regions with en-hanced sensitivity to DNase I are observed directly8). In addition, the DNase I conditions used for the

transcription factor-binding studies shown in Fig. flanking the GAL4 recognition elements, whereasthe neighboring 5S nucleosomes remain unchanged3 are similar to those in Fig. 2, lane 9 (i.e., threefold

greater than that in lane 8), suggesting that the (Fig. 3B, lane 5). These sites of hypersensitivity arenot present when GAL4-AH is absent (Fig. 3B, laneDNase I digestion profile of the reconstituted array

is produced from nucleosomal DNA, with little or 6), indicating a sequence-specific interaction be-tween GAL4-AH and the nucleosomal array to pro-no contribution from free DNA.duce a disrupted nucleosomal structure.

Analysis of Transcription Factor Binding to To investigate whether the GAL4-AH-mediatedNucleosomal Arrays chromatin remodeling leads to destabilization and

displacement of histones from the DNA, bindingHaving established the integrity of the 5S nucleo-somal array, the next step is to conduct binding reac- studies can include accessory activities that, on their

own, do not sequence-specifically interact withtions with transcription factors targeting sequencescontained within the central nucleosome. Figure 3A nucleosomal DNA, yet may play a role in reconfigur-

ing chromatin structure. An example of such an ac-provides an example of a native agarose-gel mobilityshift analysis investigating GAL4-AH binding to the tivity is the histone chaperone, nucleoplasmin,

which has been shown to stimulate transcription fac-5xGAL4/5S array. In the absence of GAL4-AH, thenucleosomal array migrates slightly faster than tor binding to nucleosome core particles by removing

H2A–H2B dimers (16) and to dissociate histonesmock-reconstituted DNA (compare Fig. 3A, lanes 1and 5). This is consistent with earlier studies demon- from core particles bound by 5 GAL4-AH dimers (17).

Interestingly, incubation of GAL4-AH and nucleo-strating that the compaction upon assembly intonucleosomes is sufficient to counter the increase in plasmin with the 5xGAL4/5S nucleosomal array

generates a DNase I digestion profile remarkablymolecular weight due to the histones (12). This assayprovides a determination of the nucleosome reconsti- similar to that for GAL4-AH alone (Fig. 3B, compare

lanes 4 and 5). However, when the samples aretution efficiency, which is nearly complete for thisexperiment, since little or no free DNA is present in passed through the oligonucleotide competition step

to remove bound factors, the GAL4 site nucleosomethe sample containing the reconstituted array.When increasing amounts of GAL4-AH are incu- exhibits much greater sensitivity to DNase I after

treatment with both GAL4-AH and nucleoplasminbated with the nucleosomal array, the array mobilityis progressively retarded (Fig. 3A, lanes 2–4). The (Fig. 3B, lane 7) than with GAL4-AH alone (Fig. 3B,

lane 8). The extensive DNase I hypersensitive sitemagnitude of the gel shift is small, yet this is ex-pected given the large size of the DNA template rela- generated by GAL4-AH and nucleoplasmin suggests

that the combined actions of these proteins suffi-tive to the DNA-binding protein. The same effect isobserved when GAL4-AH is incubated with naked ciently disrupt the underlying histones to displace

the histones. Moreover, these data illustrate thatDNA, although the amounts of protein needed toshift the DNA are less than those required for multiple proteins bound to a nucleosome-free do-

main within an array of nucleosomes (e.g., lane 4)nucleosomal DNA (Fig. 3A, lanes 6–8).To examine the specificity of transcription factor can protect the DNA from DNase I to produce a di-

gestion pattern very similar to that for the factorsbinding and to determine whether such binding al-ters nucleosomal structure, reactions are subjected bound on top of a nucleosome (e.g., lane 5). There-

fore, removal of bound factors by oligonucleotideto DNase I treatment and the digestion products areresolved on agarose gels. An example of this analysis competition may be necessary to reveal the full ex-

tent of chromatin remodeling as detected by changesfor the 5xGAL4/5S array is shown in Fig. 3B. Asexpected, DNase I readily cleaves throughout the in DNase I sensitivity in vitro.

We have also conducted studies aimed at correlat-histone-free array DNA to produce a smear of bands(Fig. 3B, lane 3). Also, the DNA-binding specificity of ing the effects of transcription factor binding to the


282 STEGER ET AL.

FIG. 3. Protein binding to the 5xGAL4/5S or HIV/5S nucleosomal array specifically remodels the central nucleosome harboringfactor-binding sites. (A) EMSA of GAL4-AH binding to the nucleosome-assembled 5xGAL4/5S array. Nucleosome-reconstituted(nucleosomal DNA) and mock-reconstituted (DNA) 5xGAL4/5S DNAs were incubated with the indicated amounts of GAL4-AH, andthe resulting complexes were resolved by agarose gel electrophoresis. (B) DNase I analysis of GAL4-AH binding to the nucleosome-assembled 5xGAL4/5S array. The DNA and nucleosomal DNA templates from A were incubated with the indicated amounts of GAL4-AH in the presence and in the absence of nucleoplasmin, and they were either treated directly with DNase I or passed through theoligonucleotide competition step to remove bound GAL4-AH prior to DNase I digestion. The digestion products were resolved byagarose gel electrophoresis. Partial digestion of 5xGAL4/5S DNA by EcoRI is shown in lane 1. Nucleosome positions are illustratedon the right. Note that the 5S nucleosome directly adjacent to the radiolabeled end of the probe DNA has been run off the gel. (C)DNase I analysis of the binding of Sp1 and NF-kB1 to the nucleosome-assembled HIV/5S array. Combinations of Sp1 and NF-kB1were incubated with the nucleosomal array in the absence and in the presence of nucleoplasmin, and they were either treated directlywith DNase I or passed through the oligo competition step to remove bound factors prior to DNase I digestion. The digestion productswere resolved by agarose gel electrophoresis. Lane 8 displays partial digestion of the HIV/5S DNA fragment by EcoRI. Nucleosomepositions and the orientation of the HIV-1 fragment within the HIV/5S template are illustrated on the right.

AID Methods A 0479 / 6714$$0479 06-11-97 23:26:33 metaas AP: Methods A


HIV/5S nucleosomal array with the in vivo chroma- is released from the tandem 5S repeats of the5xGAL4/5S array by SalI digestion, and the prod-tin structure of the integrated HIV-1 provirus (14).

In Fig. 3C, combinations of Sp1 and NF-kB1 are ucts are resolved by EMSA. In the absence of GAL4-AH, the 5-GAL4-site DNA is excised from the nucleo-incubated with the HIV/5S nucleosomal array to de-

termine whether these activators alter nuclease sen- some-reconstituted array primarily in the form of anucleosome core (Fig. 4A, lane 1). The addition ofsitivity within the HIV-1 nucleosome. Sp1 protects

a portion of the linker region in the vicinity of its GAL4-AH, followed by SalI treatment, yields com-plexes with much slower migration rates than thatbinding sites from DNase I attack (compare lane 1

with lanes 2 and 3 in Fig. 3C), while NF-kB1 yields of the core particle, indicating GAL4-AH binding(Fig. 4A, lanes 2 and 3). When nucleoplasmin isdistinct DNase I cleavage sites directly flanking both

sides of the NF-kB recognition sequences (Fig. 3C, added to the binding reactions, little change is ob-served (compare lanes 1–3 with lanes 4–6 in Fig.lanes 4 and 5). When both of these factors are added

to the array, the downstream NF-kB1-mediated 4A). To obtain an indication of the DNA state (i.e.,naked or nucleosomal) within the factor-bound com-cleavage site is reduced in magnitude, suggesting

that Sp1 binding footprints NF-kB1-mediated hy- plexes, samples are passed through the oligonucleo-tide competition step. Removal of GAL4-AH frompersensitivity (Fig. 3C, lanes 6 and 7). Interestingly,

the pattern of increased DNase I sensitivity within bound complexes in the absence of nucleoplasminregenerates the nucleosome core particle, demon-the HIV-1 nucleosome upon the binding of Sp1 and

NF-kB1 is similar to that observed on the uninduced strating that histones remain associated with theDNA under these conditions (Fig. 4A, lanes 7–9).integrated HIV-1 provirus (18). However, in contrast

to the situation involving GAL4-AH remodeling of Furthermore, the addition of nucleoplasmin in theabsence of GAL4-AH also leaves the nucleosome in-the 5xGAL4/5S array, the reconfigured HIV-1

nucleosome does not appear to be displaced. Incuba- tact (Fig. 4A, lane 10). However, the combinationof nucleoplasmin and GAL4-AH displaces histonestion of the HIV/5S nucleosomal array with Sp1, NF-

kB1, and nucleoplasmin, followed by oligonucleotide from the 5-GAL4-site DNA, since this fragment mi-grates as free DNA after oligonucleotide competitioncompetition to remove bound proteins, reveals that

the array structure reverts back to the initial state (Fig. 4A, lanes 11 and 12). Results from the nucleo-some cut-out assay agree well with the DNase I anal-without factors (see Fig. 3C, lanes 9–16). Thus,

these data suggest that the in vivo DNase I hyper- ysis. Histone displacement in the cut-out assay isobserved only under conditions yielding a dramaticsensitive sites observed for the uninduced HIV-1

provirus may result from a ternary complex con- increase in DNase I sensitivity. Furthermore, thecut-out assay is consistent with data obtained fromtaining transcription factors, histones, and DNA.

Furthermore, studies with the HIV/5S and 5xGAL4/ a mononucleosome assay investigating the dissocia-tion of histones from GAL4-AH-bound nucleosome5S array templates indicate that altered sensitivity

to DNase I can be produced by the binding of tran- core particles (Fig. 4B).scription factors directly to nucleosomes as well asby the displacement of histones.

FUTURE STUDIESDirect Determination of Histone Displacement by theNucleosome Cut-Out Assay

The extensive DNase I hypersensitive site gener- A clear avenue of work yet to be explored involvesplacing additional gene-regulatory elements into theated in the 5xGAL4/5S nucleosomal array by GAL4-

AH and nucleoplasmin is interpreted to represent a 5S array construct. The mouse mammary tumor vi-rus promoter is a prime candidate, since its chroma-nucleosome-free gap. However, an alternate possi-

bility is that histones are still associated with DNA tin organization has been well characterized underboth induced and uninduced transcriptional states.at the hypersensitive site, albeit in a severely dis-

rupted manner. The nucleosome cut-out assay is per- Moreover, gene sequences encompassing multiplenucleosomes can be inserted into the 5S array con-formed to determine the fate of histones upon tran-

scription factor binding, and the result of this assay struct to examine remodeling effects involving morethan one nucleosome. If the inserted sequences con-for the 5xGAL4/5S array is shown in Fig. 4A. In

this experiment, the central 5-GAL4-site fragment tain the start site of transcription, these constructs


284 STEGER ET AL.

FIG. 4. The concerted actions of GAL4-AH and nucleoplasmin displace histones in the 5xGAL4/5S nucleosomal array. (A) Analysisof GAL4-AH-mediated histone displacement by the nucleosome cut-out assay. Internally radiolabeled, nucleosome-reconstituted5xGAL4/5S DNA was incubated in the absence and in the presence of GAL4-AH and/or nucleoplasmin and passed through theoligonucleotide competition as indicated. Following the binding reaction and oligo competition steps, the labeled DNA fragmentharboring 5-GAL4-binding sites was released from the nucleosomal array by SalI digestion, and the digestion products were subjectedto EMSA. The migration positions for the free DNA, nucleosome core particle, and GAL4-AH-nucleosome complexes are designatedby arrows. The large fragments remaining trapped in the wells are products of incomplete SalI digestion of the nucleosomal array.(B) Analysis of GAL4-AH-mediated histone displacement by the mononucleosome assay. Nucleosome core particles containing 5-GAL4-binding sites were subjected to conditions identical to those in A.

is a recipient of a postdoctoral fellowship from the Cancer Re-can also be used as highly defined templates for RNAsearch Institute. T.O. was supported by an EMBO Long Termpolymerase II transcription assays. These experi-Fellowship. J.L.W. is a Leukemia Society Scholar.ments will help build a more complete picture of the

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Analysis of Transcription Factor-Mediated Remodeling of Nucleosomal Arrays in a Purified System

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