immunodeficiency virus type 1 during transcriptional

The EMBO Journal vol.12 no.8 pp. 3249-3259, 1993

Chromatin disruption in the promoter of humanimmunodeficiency virus type 1 during transcriptionalactivation

Eric Verdin1, Peter Paras, Jrand Carine Van Lint

Laboratory of Viral and Molecular Pathogenesis, National Institute ofNeurological Disorders and Stroke, NIH, Building 36, Room 5C22,Bethesda, MD 20892, USA'Corresponding author at: The Picower Institute for Medical Research,350 Community Drive, Manhasset, NY 11030, USA

Communicated by A.Burny

Chromatin organization of eukaryotic promoters isincreasingly recognized as an important factor in theregulation of transcription in vivo. To determine the roleof chromatin in HBIV-1 expression, we have examined thenucleosome organization of the promoter of BilV-1 underlow and high transcription rates. Independently of thecell line examined, nucleosomes are precisely positionedin the viral 5' long terminal repeat (5' LTR) anddefme two large nucleosome-free regions encompassingnt 200-450 and 610-720. A nucleosome positionedbetween these two regions, immediately after thetranscription initiation site (nuc-1), is disrupted followingTPA or TNF-x treatment. The disruption of nuc-1 fromDNA is independent of DNA replication since it iscompleted in 20 min and independent of transcriptionas it is c-amanitin insensitive. A model is proposed inwhich nuc-1 plays an organizing role in the HIV-1promoter to bring in close proximity factors bound toDNA in the two nucleosome-free regions, upstream anddownstream of the site of transcription initiation. Theseresults define chromatin as an integral component of theBiIV-1 transcriptional regulatory machinery and identifya chromatin transition associated with activation of viralgene expression.Key words: AP-1/chromatin/HIV-1/latency/nucleosome

IntroductionHuman immunodeficiency virus type 1 (HIV- 1) infection ofhumans is a progressive disease leading to severe immuno-deficiency over a period of several years. After an initialinfection, similar to many acute viral infections, an immuneresponse against the virus is generated and a longasymptomatic period follows. This asymptomatic period ischaracterized by low amounts of circulating virus and a

steady, progressive decrease in the CD4+ subset of T-lymphocytes.The replication rate of integrated HIV-1 is primarily

controlled at the level of transcription. The long terminalrepeat (LTR), present at both extremities of the integratedviral genome, contains cis-acting elements necessary fortranscription initiation (5' LTR) and for polyadenylation ofthe viral transcripts (3' LTR). The 5' LTR has beencharacterized in vitro, and binding sites for severaltranscription factors have been identified using footprinting

and gel retardation assays. They include constitutive factorssuch as COUP, API, the glucocorticoid receptor, USF,TCF- la, Spl, UBP-1/LBP-1, UBP-2 and CTF/NF1, andactivation-dependent lymphoid specific factors such as NF-xB and NFAT (reviewed in Jones, 1989; Greene, 1990;Pavlakis and Felber, 1990; Vaishnav and Wong-Staal, 1991;Gaynor, 1992).

In addition to these cellular factors, the activity of theHIV- 1 promoter is strongly dependent on the viraltransactivator tat for high activity. The R region, locatedimmediately downstream of the transcription initiation site,contains the tat responsive element (TAR), an RNA hairpinwith which tat interacts to increase the efficiency oftranscription elongation and/or initiation (reviewed in Cullen,1990; Rosen, 1991; Frankel, 1992; Karn and Graeble,1992).To obtain a full picture of HIV-1 transcriptional regulation,

the information obtained by in vitro studies, such as thedefinition of cis-acting elements and their DNA or RNAbinding proteins, should be complemented by in vivo studies.Analysis of the SV40 promoter in vivo has provided evidencefor the assembly of a higher order nucleoprotein complexthat could serve as a bridging mechanism between DNA-bound factors (Zhang and Gralla, 1989). Analysis of aregulatory region in vivo is also necessary to define whichDNA sites are occupied in vivo under various functionalstates. However, the most significant difference between invitro and in vivo analysis of a regulatory region is thepresence of chromatin in vivo. Chromatin is increasinglyrecognized as an important modulator of transcriptionalregulatory mechanisms (reviewed in Felsenfeld, 1992;Wolffe, 1992). For example, changes in chromatinorganization, such as nucleosome disruption or displacement,accompany transcriptional activation in several systems(Zaret and Yamamoto, 1984; Almer et al., 1986; Reiket al., 1991; Archer et al., 1992). Packaging of DNA ina nucleosome can prevent the binding of a DNA-bindingprotein to its recognition site (Pina et al., 1990; Taylor et al.,1991; reviewed in Hayes and Wolffe, 1992a) andnucleosome positioning can affect the function of cis-actingelements in vivo (Simpson, 1990). In addition, direct geneticevidence has been obtained in yeast for the modulating roleof chromatin on transcription: mutant yeast strains in whichthe synthesis of specific histone genes can be blocked havedemonstrated that chromatin represses the activity of severalpromoters (Han and Grunstein, 1988). Chromatin has alsobeen proposed to play an organizing role in promoter regionswhere a nucleosome could behave as a scaffolding structurenecessary to bring in close proximity regulatory factorsbound at distant sites (Elgin, 1988).To determine the role of chromatin in HIV- 1

transcriptional regulation, we have studied the nucleosomalorganization of the 5' LTR of HIV-1 integrated in severalpersistently infected cell lines. The U1 and ACH2 cell lines(Folks et al., 1987; Clouse et al., 19W-' -ntain one (ACH2)

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E.Verdin, P.Paras and C.Van Lint

or two (Ul) integrated HIV-1 genomes (Verdin, 1991) andexpress little viral RNA in basal conditions, presumably asa consequence of a block at the level of transcription(Pomerantz et al., 1990). HIV-1 transcription in these cellscan be induced up to 100-fold by treatment with cytokinesor phorbol esters (reviewed in Rozenberg and Fauci, 1990).These cells provided a well-defined system in which cis-acting elements and their cognate factors could be examinedin vivo under two dramatically different levels oftranscriptional activity.

Here, we report that the 5' region of integrated HIV-1DNA contains an array of precisely positioned nucleosomes.These nucleosomes define two large nucleosome-free regionscorresponding to the promoter region (nt 200-452) and tothe primer binding site region (nt 610-720). A nucleosomelocated in the R-U5 region in basal conditions wasspecifically disrupted following TPA or TNF-a treatment.Disruption of the R-U5 nucleosome was independent oftranscription and independent of DNA replication.

ResultsNucleosomes are precisely positioned in the 5' LTR ofHIV-1To determine the nucleosomal organization of the 5' regionof integrated HIV-1, nuclei from HIV-1 persistently infectedcell lines were prepared and treated with DNase I ormicrococcal nuclease. After digestion of nuclei, DNA wasextracted, purified and analyzed by indirect end-labeling(Wu, 1989) and Southern blotting as previously described(Verdin, 1991). In this and the following experiments, cellswere examined under two conditions: treated or not treatedwith TPA. When treated, cells were incubated in thepresence of TPA for at least 12 h and the efficiency of viralinduction was monitored by measuring the secreted p24antigen in the medium (Verdin, 1991). Previous experimentshave shown that, following TPA treatment, viral transcriptsslowly accumulate to reach a maximum after 24 h(Pomerantz et al., 1990). After DNase I digestion of nuclei,three regions were found to be preferentially digested in the5' region of the HIV-1 genome as previously reported(Verdin, 1991). Two of these regions, called hypersensitivesites 2 and 3 (HS 2, 3), are associated with the promoterin the U3 region (HS2 encompassing nt 223-325; HS3: nt390-449) (Figure 1A and B; lanes 18-19). Anotherhypersensitive site, HS4, was found immediatelydownstream of the U5 region (HS4: nt 656-720) in bothcell lines (Figure 1). After induction with TPA, a markedincrease in sensitivity to DNase I was noted between HS3and HS4 (nt 449-656) (indicated by thick arrows inFigure 1A and B), whereas other hypersensitive sites wereunchanged (Figure LA and B; lanes 20-21). Controldigestion of naked genomic DNA in vitro with DNase Ishowed no preferential cutting as previously described(Verdin, 1991). To confirm that these hypersensitive regionswere not an artefact of nuclei preparation and could also bedetected in vivo, the two cell lines were also digested withDNase I after permeabilization with lysolecithin (Pfeifer andRiggs, 1991). The same three hypersensitive sites, HS 2,3 and 4, were observed; however, a high background of non-specific digestion was noted and this approach was notexplored further.To map nucleosome boundaries in the 5' LTR, we have

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Fig. 1. Micrococcal nuclease and DNase I digestion of HIV-1chromatin. Nuclei from Ul (A) or ACH2 cells (B) untreated or treatedwith TPA for 12 h were digested with micrococcal nuclease or DNaseI and examined by indirect end-labeling after PstI digestion in vitro.Naked DNA purified from the two cell lines was digested in vitro withthe same nucleases as a control. The following concentrations ofmicrococcal nuclease were used: lanes 1, 6, 11: 0 U/ml, lanes2, 7: 0.015 U/ml, lanes 3, 8: 0.03 U/ml, lanes 4, 9: 0.06 U/ml, lanes5, 10: 0.12 U/ml, lanes 12: 0.00016 U/ml, lanes 13:0.0008 U/ml, lanes 14: 0.004 U/ml, lanes 15: 0.02 U/ml, lanes 16:0.1 U/ml, lanes 17: 0.5 U/ml. DNase I was used at 0 U/ml: lanes 18,20 and 20 U/ml: lanes 19, 21. Molecular weight markers are a doubledigest of HIV-LAI DNA by PstI (nt 1415) and EcoRV (nt 112,marker a), Hpal (nt 309, marker b), AJM (nt 517, marker c), SacI(nt 678, marker d), HgaI (nt 792, marker e), AccI (nt 959, marker f),Hindm (nt 1085, marker g). HS refers to hypersensitive site.

used micrococcal nuclease. This enzyme preferentially cutsDNA present in nucleosome-free regions in chromatin aswell as DNA present in linker domains betweennucleosomes. Such a digestion could, in theory, generatetwo different patterns. If nucleosomes were preciselypositioned in all integrated copies, the digestion in similarlypositioned linker regions would yield discrete bands. If, onthe contrary, nucleosomes were randomly distributed,digestion in linker regions would occur randomly and asmear of digestion products spanning the region would beobserved. Digestion of nuclei from untreated ACH2 and Ulcell lines revealed a discrete banding pattern (indicated bysmall arrows in Figure IA and B; lanes 1-5), a resultconsistent with precise nucleosome positioning. Digestion

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Fig. 2. Model for the chromatin organization of the 5' LTR of HIV-1. Sites of cutting by DNase I and micrococcal nuclease in purified nuclei are

indicated by solid bars and are aligned with cis-acting elements in the 5' LTR. A tentative assignment of nucleosome positions in this region basedon nuclease digestion is shown above.

of naked genomic DNA in vitro under the same conditionsof buffer and enzyme concentrations showed no specificpattern of digestion (Figure LA and B; lanes 11-17),supporting the hypothesis that the pattern observed was

secondary to chromatin organization. When the same regionwas examined after TPA treatment, similar results were

obtained (Figure IA and B; lanes 6-10) except that, as

described for DNase I, the R-U5 region becamehypersensitive to digestion (Figure IA and B; lanes 6-10,indicated by thick arrows). Similar digestions with DNaseI and micrococcal nuclease were performed using two otherHIV-l chronically infected cell lines, the 8E5 (Folks et al.,1986) and the OM10. 1 cell lines (Butera et al., 1991), andshowed essentially the same results (Verdin, 1991; E.Verdin,unpublished observations).

Results from several experiments were averaged and a

tentative map of nucleosome positioning in the 5' region ofthe HIV-1 genome was established (Figure 2). A singlenucleosome (nuc-0) is present in the U3 region,encompassing nt 40-200, whereas the rest of the U3 region(nt 200-452) is open and highly accessible to both DNaseI (HS2 and HS3) and micrococcal nuclease. The R-U5 regionis resistant to nuclease digestion in basal conditions, butbecomes sensitive to both nucleases after TPA treatment(wider boxes in Figure 2). This region spans nt 452 -596,or 144 nucleotides, which corresponds approximately to thelength ofDNA protected by a nucleosome core. It is possiblethat a nucleosome is present in this location in basalconditions and disrupted upon activation of HIV-1transcription. The DNA domain separating this nucleosome(nuc-1) from the next (nuc-2) spans 124 nt, which isunusually large for a linker region and may account for itshypersensitivity to DNase I (HS4). The region downstreamcontains three precisely positioned nucleosomes, referred toas nuc-2, nuc-3 and nuc-4 (Figure 2).

Restriction enzyme digestion of chromatin confirmsthe proposed nucleosome positioningTo confirm our model for nucleosome positioning in the 5'LTR, we have used restriction endonucleases which do notdigest nucleosomal DNA, but readily cut linker andnucleosome-free DNA. The efficiency of digestion ofchromatin by a restriction enzyme depends on severalvariables. These include: (i) the accessibility of the DNA(i.e. nucleosomal or naked); (ii) the activity of the enzymein the digestion buffer; (iii) the size of the enzyme and itsability to enter the nuclei; (iv) the stability of the restrictionenzyme in the presence of proteases and/or other factorspresent in nuclei under digestion conditions; and (v) thepresence of DNA-binding proteins competing for the same

site. As described for DNase I and micrococcal nuclease,nuclei from untreated and TPA-treated ACH2 and Ul cellswere prepared, digested with 16 restriction endonucleasescutting at 25 distinct sites in the 5' region of the HIV-1genome and analyzed by indirect end-labeling. The efficiencyof digestion at each site was plotted as a function of itsposition in the genome (Figure 3). The proposed assignmentof nucleosomes derived from Figure 2 is shown for reference(Figure 3). Two large peaks of increased enzyme digestion(from D to K and from 0 to T) were found to coincideprecisely with the two regions predicted to be nucleosome-free on the basis of digestion with micrococcal nuclease andDNase I (Figure 3). Similar results were obtained with theUl cell line (data not shown). After TPA treatment, thepattern of digestion by most of these enzymes was essentiallythe same, except for the region encompassing R-U5. Forthree enzymes (AflII: nt 517, HindUl: nt 531, Hinfl: nt 577indicated by an asterisk), a significant increase in digestionwas noted following TPA treatment (Figure 4). Sincedigestion by several enzymes located outside of this regionwas either unchanged (AvaL: nt 295, MspI: nt 309, AlwNI:

3251

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nt 421; Figure 3A and B) or decreased (Pvull: nt 435, HaeI:nt 641) after TPA treatment, the increased digestion isprobably reflective of a true increase in accessibility(Figure 4). In addition, the Hinfl digestion provided an

excellent internal control since this enzyme was shown tocut at another site in the HIV-1 genome (nt 695, indicatedby an open circle) at the same rate, independently of TPAtreatment (Figure 4A and B).

High-resolution analysis of the R-U5 regionChanges in DNA accessibility for nucleases in the R-U5region following induction of viral expression could resultfrom the disruption or displacement of a positionednucleosome, from the displacement of several DNA-bindingfactors from the region or from the disruption of a higherorder structure such as a folded DNA loop. Because of thelocation of the R-U5 region next to the site of transcriptioninitiation, the changes observed could reflect an importantmechanism in the transcriptional activation of the HIV-1promoter in vivo. To define these changes further, the regionwas examined at high resolution using ligation-mediated PCR(LMPCR) after modification ofDNA in purified nuclei withmicrococcal nuclease, DNase I and dimethylsulfate (DMS).

Fig. 3. Digestion of HIV-l chromatin with restriction enzymes.Untreated ACH2 nuclei were digested in vitro with 16 differentrestriction enzymes. After purification of DNA and secondaryrestriction in vitro with PstI, DNA samples were analyzed by indirectend-labeling and Southern blotting. The efficiency of cutting at a givensite was assessed by densitometric scanning of the band resulting fromthe digestion and is presented in optical density (OD) arbitrary units.The x-axis shows the relative position of each of the restriction siteson the HIV-1 genome and aligned with it is a model of nucleosomepositioning derived from Figure 2. The following enzymes were used:A = EcoRV (nt 35), B = Sau96I (nt 99), C = EcoRV (nt 114),D = DrafI (nt 212), E = Sau96I (nt 293), F = AvaI (nt 295),G = MspI (nt 309), H = ScaI (nt 315), I = BanHl (nt 413),J = AlwNI (nt 421), K = PvulI (nt 435), L = Afll (nt 517),M = HinduI (nt 531), N = Hinfl (nt 577), 0 = HaeH (nt 641),P = Sau96I (nt 652), Q = Sacl (nt 682), R = Banll (nt 682),S = Hinfl (nt 695), T = Bsp5OI (nt 713), U = ClaI (nt 831),V = Hinfl (nt 917), W = HindIll (nt 1085), X = AlwNI and Pvul(nt 1147).

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Micrococcal nuclease. Micrococcal nuclease digestion ofuntreated nuclei revealed the presence of a 145 nt footprintextending from nt 465 to nt 610 when compared with nakedDNA digested in vitro (Figure 5A; lanes 1-4 and 7-10).Following TPA treatment, this region became sensitive todigestion and was digested by micrococcal nuclease in amanner undistinguishable from naked DNA (Figure SA;compare lanes 5-6 with 1-2, lanes 11-12 with 7-8).

Dimethylsulfate. The region was further studied after DMStreatment ofDNA in vivo. DMS methylates guanosine and,to a lesser degree, adenosine residues in DNA in living cells,except if these residues are engaged in a close interactionwith a DNA-binding factor. Remarkably, the packaging ofDNA in a histone octamer does not interfere with itsmethylation by DMS and nucleosomal DNA is modified invivo in a manner undistinguishable from naked DNA. Intactcells, treated or not treated with TPA, were incubated briefly

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Fig. 4. Effect of TPA treatment on restriction enzyme digestion of chromatin. Nuclei from untreated (-) and TPA-treated (+) ACH2 (panel A) andUl (panel B) cells were purified and digested with the indicated restriction enzymes. After purification and restriction digestion in vitro with PstI,samples were examined using indirect end-labeling and Southern blotting. Induction refers to the ratio between TPA-treated and untreated samplesafter scanning the corresponding autoradiographs. Molecular weight markers are the same as in Figure 1. The asterisk (*) refers to the Hinfl site atnt 577, whereas the open circle (0) refers to the Hinfl site at nt 695.

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Fig. 5. High-resolution analysis of the chromatin organization in the R-U5 region. DNA samples were treated in vivo or in vitro with DNase I,micrococcal nuclease or DMS, amplified using LMPCR and the amplified products analyzed on 6% denaturing polyacrylamide gels. (A) Micrococcalnuclease. Two different concentrations of micrococcal nuclease were used for each condition: lane 1: 0.1 U/ml, lane 2: 0.02 U/ml, lane 3: 0.1 U/ml,lane 4: 0.04 U/ml, lane 5: 0.1 U/ml, lane 6: 0.04 U/ml, lane 7: 0.04 U/ml, lane 8: 0.02 U/mil, lane 9:0.015 U/ml, lane 10: 0.03 U/ml, lane 11:0.015 U/ml, lane 12: 0.03 U/mil. Genomic DNA modified in vitro according to Maxam-Gilbert sequencing reaction was also amplified and used as

a size marker. (B) DMS. Living cells, untreated or TPA treated, or genomic DNA were treated with DMS and then subjected to piperidine cleavagein vitro, followed by LMPCR. The position of the footprint observed with micrococcal nuclease in panel A is indicated on the right.Maxam-Gilbert sequencing products were also amplified and used as size markers. (C) DNase I. Nuclei from untreated or TPA-treated cells were

digested with DNase I at 20 U/mi (lanes 1 and 2) and compared with naked genomic DNA digested in vitro with 1 U/mi (lane 3). Samples from a

micrococcal nuclease digestion were analyzed and run in parallel with these samples as a reference. Micrococcal nuclease concentrations were: lanes4, 6: 0.12 U/ml; lanes 5, 7: 0.06 U/ml; lane 8: 0.1 U/ml; lane 9: 0.02 U/ml. The position of the putative nucleosome is indicated. Lanes 1 and 2were densitometrically scanned and the result of the subtraction of plots of lane 1 from lane 2 is shown aligned with the digestion products(lane 2-lane 1). Maxam-Gilbert sequencing reactions were run on the same gel and used as size markers.

in the presence of DMS, the reaction was stopped, DNApurified and treated with piperidine to cleave DNA at thesite of methylation by DMS. Naked DNA was treatedsimilarly in parallel. DNA samples were amplified byLMPCR and examined on sequencing gels (Figure SB).These experiments indicated no difference between DNAmodified in vitro or in vivo and no difference between TPA-treated and untreated cells (Figure 5B). This result supportedthe hypothesis that, in the absence of TPA, this region isoccupied by a nucleosome rather than by several contiguousDNA-binding proteins.

DNase I. Digestion of nucleosomal DNA with DNase I invitro shows a distinct pattern of decreased digestion with a

periodicity of 10-11 nt when compared with naked DNA.This periodic decrease in digestion is thought to reflect theperiodic decrease in accessibility of DNA wrapped aroundthe histone octamer (Hayes et al., 1990). DNA samplesobtained after DNase I digestion of naked DNA, or of nucleifrom untreated and TPA-treated cells, were analyzed afterLMPCR amplification of the digestion products. DNase Idigestion of chromatin in TPA-treated cells and of nakedDNA yielded essentially the same pattern of fragments,confirming the results obtained with micrococcal nuclease(Figure 5C; compare lanes 1 and 3). In contrast, severaldifferences were noted between DNA from untreated and

TPA-treated cells (Figure 5C; lanes 1 and 2). These twolanes were densitometrically scanned and the resulting plotssubtracted in order to better visualize changes(lane 2-lane 1). Several major negative peaks were notedin the region where a large footprint had been noted withmicrococcal nuclease (Figure 5C). A negative deflectionindicated a relative protection in untreated cells versus TPA-treated cells and several of these peaks were found to beseparated by an integral repeat of 10-11 bp. However, notall expected peaks were present and this could reflect thepresence of a DNA-binding factor in the region afterinduction with TPA or the fact that the region was poorlydigested by DNase I. It is also possible that the precisionof positioning of nuc- I is not one nucleotide, but rather twoor three nucleotides, a property which would obscure the10 bp repeat. This periodic protection from DNase I was

only observed in the region encompassing the micrococcalnuclease footprint (nt 465 -610) (Figure 5C). DNAdownstream of the nucleosome was digested to the same

extent before and after TPA, as reflected by the plot(lane 2-lane 1 = 0). DNA upstream of nt 465 was more

sensitive to DNase I digestion in untreated than in TPA-treated cells (Figure 5C). The same hypersensitivity was alsonoted in this region with micrococcal nuclease (Figure 5Aand C). It is possible that this hypersensitivity is secondaryto the presence of localized melting of the DNA double helix

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at the transcription start as single-stranded DNA is markedlymore sensitive to micrococcal nuclease digestion than double-stranded DNA (Drew, 1984; Zhang and Gralla, 1989).Thus, high-resolution analysis of the R-U5 region in the

5' LTR of HIV-1 is consistent with the presence of aprecisely positioned nucleosome encompassing nt 465-610in untreated cells. Following TPA treatment, this nucleosomeis disrupted or displaced from the DNA and DNA is digestedsimilarly to naked DNA.

Disruption of nuc-l is rapid and independent of DNAreplicationEvidence has been presented that nucleosome disruption andreassembly can occur rapidly and independently of DNAreplication (Zaret and Yamamoto, 1984; Richard-Foy andHager, 1987; Reik et al., 1991; Schmid et al., 1992). Toaddress this question in our system, we have examined thekinetics of the disruption of nuc-1. ACH2 cells were treatedwith TPA or TNF-a for variable periods of time, rapidlycooled on ice and nuclei were prepared. Nuclei were digestedas described above with AJM or Hinfl and processed forindirect end-labeling. An increase in accessibility of thenucleosome region was noted for both enzymes as rapidlyas 20 min following treatment with either TPA or TNF-cx(Figure 6A and B). The increase in accessibility was specificfor this region as another cutting site for Hinfl, at nt 695,was unaffected by either treatment. The same experimentwas performed with the U1 cells and similar results werenoted (data not shown). Given the kinetics of replication ofthese cells (doubling time of - 24 h) and the fact that thecultures were not synchronized, these results confirm thatDNA replication is not necessary for the disruption of nuc-1.

Disruption of nuc- 1 is insensitive to a-amanitinTranscription of a nucleosomal template by RNA polymeraseII could potentially disrupt its nucleosomal organization andseveral studies have suggested that an altered chromatinstructure exists on transcribed sequences (Nacheva et al.,1989; Chen et al., 1990; Lee and Garrard, 1991; Clark andFelsenfeld, 1992; reviewed in Kornberg and Lorch, 1991).To examine the possible role of transcription in the disruptionof nuc-1, cells were pretreated for 1 h with a-amanitin, anRNA polymerase II inhibitor, to block transcription,followed by TPA treatment for 1 h and the R-U5 region wasthen examined. To confim the inhibition ofRNA synthesis,RNA was extracted from treated and untreated cells, andhybridized with an HIV-1-specific probe. TPA treatmentresulted in a > 100-fold increase in virus-specific RNA(Figure 7B). This increase was accompanied, as expected,by nucleosome disruption (Figure 7C and D). In cells thatwere pretreated with ai-amanitin, the induction of viralexpression was suppressed by >90% (Figure 7B); however,this inhibition had no effect on the disruption of thenucleosome which occurred to the same extent as in theabsence of oa-amanitin (Figure 7C and D). These resultsprove that the bulk of the transcriptional increase observedafter TPA treatment is not necessary for the disruption ofnuc-1, but do not rule out the role of transcription completelysince residual transcription occurring after a-amanitintreatment could be playing a significant role.

DiscussionWe have examined the chromatin organization of integratedHIV-1 in chronically infected cell lines. Precisely positioned3254

nucleosomes and intervening nucleosome-free regions arefound in the 5' LTR region where viral cis-acting elementsare located. A large nucleosome-free region is foundassociated with the enhancer/promoter region (nt 200 to nt452-465) and with a region downstream of the site oftranscription initiation (nt 610-720) (Figure 2). Twonucleosomes are precisely positioned in the 5' LTR,encompassing nt 40-200 (nuc-0) and nt 465 -610 (nuc- 1).Following transcriptional activation, nuc-1 is specifically andrapidly disrupted, and the underlying DNA is digested ina manner similar to naked DNA.The presence of a nucleosome-free region preceding the

site of initiation of transcription has been described for manydifferent promoters (Gross and Garrard, 1988). It isgenerally accepted that the binding of transcription factorsto DNA in a promoter region prevents its incorporation intoa nucleosomal structure. The indirect end-labeling techniqueused here did not provide a resolution sufficient for theidentification of the factors bound in the enhancer andpromoter regions in vivo. However, since the region isnucleosome-free, all factors previously found to bind in vitro(reviewed in Gaynor, 1992) can in theory compete for theirrespective sites in vivo.Another nucleosome-free region was found downstream

of the initiation site, covering nt 610-720. No regulatoryfunction has been reported for this region of the viralgenome. However, because open chromatin structuresfrequently reflect the association of DNA-binding factorswith DNA in vivo, we have examined this region for potentialprotein binding sites. In vitro footprinting analysis of thisregion with nuclear extracts from lymphoid cell linesidentified binding sites for transcription factors AP3, Spiand for a new factor that we have called DownstreamBinding Factor (DBF) (Figure 2). Using genomicfootprinting with DMS, we have also found that the DBFand Spi sites are occupied in vivo in the U1 and ACH2 celllines (Elkharroubi and Verdin, submitted).

In basal conditions, a nucleosome referred to as nuc-0encompasses nt 40-200 and contains a portion of thenegative regulatory element (NRE), a silencer of the viralpromoter (Rosen et al., 1985) (Figure 2). Several bindingsites for factors have been described in this region in vitro,including AP-1, NFAT, COUP and the glucocorticoidreceptor (reviewed in Gaynor, 1992). However, packagingof DNA in a nucleosome distorts the helical path of DNA(Hayes et al., 1990) and consequently the recognition of aDNA-binding site by its cognate factor can be impaired(Taylor et al., 1991; Hayes and Wolffe, 1992b). For thisreason, the biological significance of the binding sitesdescribed in this region in vitro should be re-evaluated inthe context of chromatin structure.A second nucleosome in the 5' LTR (nuc- 1) is positioned

between nt 465 and nt 610 in basal conditions (Figure 2).This nucleosome is rapidly and specifically disruptedfollowing treatment with TPA or TNF-a, two agents knownto increase HIV-1 transcription in these cells. The specificityof this disruption is established by the observation that othernucleosomes in the region (nuc-0, 2, 3, 4) are not affectedby TPA treatment. A time course experiment indicates thatthe disruption is rapid and essentially completed in 1 h. Sincean increase in viral transcripts is only observed 6-8 h afterTPA treatment (Pomerantz et al., 1990), this result impliesthat nucleosome disruption precedes transcriptionalactivation. Further proof of the independence of disruption

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of nuc-1 from transcriptional activation is the observationthat disruption still occurred when transcription was blockedby cx-amanitin.

Chromatin remodeling during transcriptional activation hasbeen reported in other inducible promoters: the yeast PHO5gene (Almer et al., 1986), the LTR of mouse mammarytumor virus (MMTV) (Zaret and Yamamoto, 1984; Bresnicket al., 1992) and several heat shock protein (hsp) genes inDrosophila (Cartwright and Elgin, 1986). These genes havein common with HIV- 1 a low basal transcription rate anda high increase in transcription rate in response to activatingfactors. It has been proposed that the presence of a positionednucleosome, or of histone Hi, in the promoter of these genes

under basal conditions blocks access to DNA fortranscription factors that are crucial for promoter activity.Transcriptional activation of these genes is preceded or

accompanied by the disruption of several nucleosomes(Schmid et al., 1992), or of histone HI only (Bresnick et al.,1992), allowing access to DNA for DNA-binding factors.Experiments in yeast using conditional mutants, in whichthe expression of selected histones can be suppressed, haveconfirmed these predictions and proved that histones exerta repressive activity on the expression of the PHO5 genein basal conditions (Han and Grunstein, 1988).The presence of a nucleosome in basal conditions in the

HIV- 1 5' LTR and its rapid disruption upon transcriptionalactivation raise several interesting possibilities for the

transcriptional regulation of HIV-1. In basal conditions, thisnucleosome is surrounded by two large domains ofnucleosome-free DNA where several sites for DNA-bindingfactors are located. This organization is compatible with a

model first proposed for the Drosophila heat shock protein(hsp) 26 promoter where a nucleosome acts as a scaffoldingstructure bringing in close apposition two regions wheretranscription factors are bound (Elgin, 1988). Thenucleosome creates a static loop which facilitates theinteraction between distantly bound factors. The functionalimportance of a nucleosome-mediated loop in thetranscription of a promoter has recently been illustrated bystudies of the vitellogenin BI promoter (Schild et al., 1993).In this system, an estrogen-responsive element is separatedfrom other DNA-binding proteins in the promoter by a 160bp interval where a nucleosome is positioned. Nucleosomeassembly of the vitellogenin B1 promoter resulted in a

potentiation of transcription in vitro (Schild et al., 1993).In the case of the HIV-1 promoter, it is interesting to notethat Spi binding sites are present in the two nucleosome-free regions that surround nuc-1, upstream (Jones et al.,1986) and downstream, associated with HS4 (Elkharroubiand Verdin, submitted) (Figure 2). Since Spl can mediatethe formation of a DNA loop between distant binding sitesin vitro (Mastrangelo et al., 1991; Su et al., 1991), it ispossible that the interaction between SpI proteins boundupstream and downstream of nuc-1 could be facilitated by

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A


Bo


0

, 0 ..

Fig. 7. Disruption of the R-U5 nucleosome occurs in the presence of a-amanitin. ACH2 cells pretreated or not with a-amanitin (5 yg/ml) for 1 hwere induced with TPA for 4 h. RNA was purified and analyzed on an agarose gel, and examined after ethidium bromide staining (panel A). RNAwas also loaded on a formaldehyde/agarose gel and HIV-1-specific transcripts were detected after transfer and hybridization according to theNorthern blotting procedure. The three major transcripts characteristic of productive HIV-1 infection are detected (panel B). Nuclei from these cellswere prepared and the accessibility of the R-U5 region assessed using AJfl and Hinfl digestion as described before (panel C). Panel D shows thequantitation of these digestions after densitometric scanning of autoradiograms.

the incorporation of the intervening DNA into nuc-l(Figure 8). The interaction between Spi proteins boundupstream and downstream of nuc- 1 could in turn stabilizethe interaction of nuc-1 with DNA. Such a configuration ofthe HIV-1 promoter brings in close proximity other factorsbound to DNA upstream and downstream of nuc- 1,facilitating their interaction (Figure 8). Since nuc- 1 is onlypresent in basal condition when the promoter is repressed,it is reasonable to hypothesize that the net result of thisinteraction is predominantly negative.The presence of a nucleosome downstream of the site of

transcription initiation (nt 455) in the HIV-1 promoter, suchas nuc- 1, could interfere with transcription initiation orelongation. Indeed, packaging ofDNA in a nucleosome canalter its recognition by transcription factors (Lorch et al.,1987; Morse, 1989; reviewed in Kornberg and Lorch, 1991)and several factors, such as LBP-1, UBP-1 and CTF/NF1(Garcia et al., 1987; Jones et al., 1988; Malim et al., 1989)are known to bind close to or in the DNA domain packagedinto nuc-l.Nucleosomes can also inhibit elongation by RNA

polymerase II (Izban and Luse, 1991) and RNA polymeraseIII (Morse, 1989). In the case of pol II, this inhibition was

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secondary to an enhancement of DNA sequence-specificpausing on chromatin versus naked templates (Izban andLuse, 1991). Similarly, the presence of nuc-1 in the HIV-1leader can, in theory, accentuate a natural pausing site forpol II in this region and interfere with transcriptionelongation. Short attenuated transcripts have been detectedin the leader region of the HIV- 1 promoter in vivo in theabsence of the viral transactivator tat (Kao et al., 1987;Laspia, 1990; Ratnasabapathy et al., 1990; Feinberg et al.,1991, Kessler and Mathews, 1992). The HIV-1 protein tatalleviates this block and allows transcriptional elongation toproceed (Kao et al., 1987; Laspia, 1990; Feinberg et al.,1991; Kessler and Mathews, 1992).The mechanism underlying low-level expression in the U1

and ACH2 cell lines is not fully understood. This form oflow-level expression has been referred to as post-integrationlatency (Garcia-Blanco and Cullen, 1991). The HIV-1infectious cycle appears to be blocked at an early stage inthese cells and their pattern ofmRNA expression is similarto that observed with rev- mutant HIV-1 since onlymultiply spliced transcripts are detected in basal conditions(Pomerantz et al., 1990). Rev protein requires multi-merization for activity and it has been suggested that a

): le-! "', !.


threshold intracellular concentration of rev is necessary formultimerization of rev to occur efficiently and for itsbiological activity (Malim and Cullen, 1991; Pomerantzet al., 1992). The primary cause for a low rev concentrationappears to be a transcriptional block in HIV-1 expression(Pomerantz et al., 1990). The strong dependency of theHIV-1 promoter on cellular activation signals, mediated inpart by NF-xB binding sites, has been proposed as themechanism underlying the activation of HIV-1 expressionin ACH2 and Ul cells following TPA or TNF-ov treatment,as both of these agents increase NF-xB activity (Griffinet al., 1989; Osborn et al., 1989). However, the absenceof NF-xB in basal conditions is not sufficient to fully explainlatency. Indeed, if this were true, HIV-1 mutants with noNF-xB binding sites in their LTR would be expected toalways be latent. The fact that this has not been observed(Leonard et al., 1989; Ross et al., 1991) suggests thatanother factor is responsible for suppressing basal HIV-1transcription in these cells. Results presented here areconsistent with a model in which the presence of nuc-1 isthe primary cause for the suppression of transcription in theHIV-1 promoter in basal conditions, either by inhibitingtranscription initiation or by accentuating a natural pausingsite for the polymerase in the R region of the viral LTR.According to this model, activation of HIV- 1 expression inthese cell lines is a bimodal phenomenon consisting of boththe disruption of nuc-1, allowing transcription initiation orelongation, and the activation of NF-xB, resulting in anincrease in initiation efficiency. This model is supported bystudies on the mechanism of HIV-1 induction by UV lightand DNA-damaging agents where a chromatindecondensation step has been proposed as the triggeringmechanism during activation (Valerie and Rosenberg, 1990).The mechanism for the disruption of nuc-1 is at present

unclear. The interaction of most nucleosomes with DNA isinherently stable and the translational position of thenucleosome is dictated by the DNA sequence (Fitzgerald andSimpson, 1985; Drew and Calladine, 1987). Directdestabilization by a DNA-binding protein has been proposedas a disruption mechanism in several systems in vivo (Fascheret al., 1990; Reik et al., 1991; Archer et al., 1992). In vitro,binding of the yeast GAL4 protein to its cognate siteincorporated into a nucleosome resulted in the formation ofan unstable ternary complex composed of the DNA, thehistone octamer and GAL4. This complex could dissociatein either the original nucleosome core or GALA bound tonaked DNA (Workman and Kingston, 1992). Similarly, inthe HIV-1 promoter, as AP-1 is a TPA-inducible factor andas three AP-1 binding sites are located in the DNA regionpackaged in nuc-1 (Elkharroubi and Verdin, submitted; seealso Figures 2 and 8), the AP1 factor is a primary suspectin the disruption of nuc-1 . In addition, acetylation of the corehistone tails has been shown to destabilize the nucleosomecore from DNA (Oliva et al., 1990; Walker et al., 1990)and could play a role in facilitating transcription factorinvasion of a nucleosomal region (Lee et al., 1993).The observations described here establish the basis for an

integrated understanding of HIV-1 transcriptional regulationin vivo. The mechanism of disruption of nuc-1, and itspotential role in modulating transcription initiation orelongation, is being examined by reconstitution in vitro ofchromatin templates containing the HIV- 1 promoter. Thesestudies should contribute to our understanding of HIV-1regulation and ultimately AIDS pathogenesis.

TPATNF-M

5,

Fig. 8. Hypothetical model for the chromatin organization of the 5'portion of the HIV-1 genome. A region of the HIV-1 genomeextending from nt -340 to nt - 1100 is depicted as a black ribbon.DNA-binding factors and three nucleosomes interacting with thisregion are drawn at the appropriate relative position on the viralgenome. Wrapping of DNA around nuc-l positions two Spl bindingsites located in the U3 region in front of 2 Spl binding sites describedin the leader sequence (Elkharroubi and Verdin, submitted) with whichthey could interact. Nucleosomes are drawn to scale with respect tothe DNA. Transcription factors and RNA polymerase II are drawn totheir approximate size with respect to nucleosomes and DNA. TBPrefers to TATA-binding protein, LBP refers to several leader bindingproteins that generate a large footprint around the transcription start inin vitro footprinting studies (Garcia et al., 1987; Jones et al., 1988;Maim et al., 1989).

Materials and methodsCell linesThe ACH2 and Ul cell lines were obtained from the AIDS Research andReference Reagent Program (NLAID, NIH, Bethesda, MD). Cells weregrown in RPMI (Gibco/BRL) 10% fetal calf serum (Hyclone), supplementedwith 50 U/ml of penicillin, 50 ytg/ml of streptomycin and 2 mM glutamineat 37°C in a 95% air/5% CO2 atmosphere and were maintained at a densityof 0.25-1 x 106 cells/ml.

Nuclease digestion of purified nucleiExponentially growing cells were harvested by centrifugation at 1000 r.p.m.(GPR table top centrifuge, Beckman) for 10 min at 4°C and washed twicewith ice-cold phosphate-buffered saline (PBS). All subsequent operationswere performed on ice with precooled buffers. Cells were counted andresuspended at 25 x 106 cells/ml in buffer A [10 mM Tris (pH 7.4),10 mM NaCl, 3 mM MgCl2, 0.3 M sucrose] and incubated on ice for 5min. An equal volume of buffer A/0.2% NP40 was added and the cellswere incubated for another 5 min with intermittent mixing. Nuclei werepelleted at 1000 r.p.m. for 10 min and resuspended at 108 nuclei/mi in oneof three buffers depending on the nuclease used for digestion: buffer A forDNase I, buffer A supplemented with 10 mM CaC12 for micrococcalnuclease, buffer B [10 mM Tris (pH 7.9), 10 mM MgCl2, 50 mM NaCl,1 mM dithiothreitol (DTT), 100 sg/ml bovine serum albumin (BSA),0.1 mM PMSF] for restriction enzymes. Nuclei were digested for 10 minon ice with DNase I, for 20 min at 22°C for micrococcal nuclease andfor 20 min at 37°C for restriction enzymes. Digestion reactions were stoppedby adding 1 vol. of 2 x proteinase K buffer [100 mM Tris (pH 7.5), 200mM NaCl, 2 mM EDTA, 1% SDS] and mixing. Samples were solubilizedfor 1 h at 55°C, proteinase K was added at 200 Ag/mi and the digestionallowed to continue overnight at 550C. RNase was added at a finalconcentration of 50 isg/ml for 1 h at 370C. Samples were extracted threetimes with phenol, three times with chloroform/isoamyl alcohol (24:1) andprecipitated with ethanol. DNA was resuspended in sterile water and itsconcentration estimated by measuring the absorbance at 260 nm.

Nuclease treatment of naked DNADNA from exponentially growing untreated cells was purified after an

overnight digestion with 200 Ag/ml of proteinase K in proteinase K buffer.After three phenol extractions and three chloroform/isoamyl alcohol

3257


extractions, DNA was ethanol precipitated and resuspendedin sterile water.DNA was digested for 10min on ice for DNase I or 20 min at 22°C formicrococcal nuclease with DNA at a concentration of 0.3 mg/ml. Reactionswere stopped by the addition of proteinase K buffer and processed asdescribed for the nuclei after DNase I treatment.

Southern blottingPurified DNA (30 jig) was digested with PstI and the fragments generatedwere separated by electrophoresis in 0.8 or 1.5% agarose gels in Tris-boratebuffer at 1.5 V/cm. Size markers were described previously (Verdin, 1991)and were electrophoresed along with the samples. Agarose gels wereincubated 2 x 20 min in denaturing solution (1.5 M NaCl, 0.5 M NaOH),2 x 20 min in neutralizing solution [1.5 M NaCl, 0.5 M Tris (pH 7.2),1 mM EDTA] and transferred overnight by capillarity in 20 x SSPE [3M NaCl, 0.2 M NaH2PO4, 20 mM EDTA (pH 7.4)] to nylon membranes(N-Hybond, Amersham). DNA was cross-linked to nylon membranes byexposure to UV light (UV Stratalinker 1800, Stratagene), washed for 20min in 2 x SSPE and prehybridized for1-2 h at 420C in hybridizationbuffer [50% formamide, 3.6 x SSPE, 1% SDS, 10% dextran sulfate,5 x Denhardt's (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% BSA),0.1 mg/ml sonicated herring sperm DNA]. Probe A spanning nucleotides643 -1415 (HIVLAI) was prepared and labeled as previously reported(Verdin, 1991). The denatured DNA probe was added to the prehybridizationbuffer and allowed to hybridize for at least 16 h at 42°C. Membranes werewashed for 2 x 20 min in 2 x SSPE/0.1% SDS, 2 x 20 min in0.2 x SSPE/0.1 % SDS at room temperature, followed by 30 min at 650Cin 0.2 x SSPE/0.1 % SDS. Autoradiographic exposures were carried outfor 1-5 days at -700C with two intensifying screens.

RNA purification and Northern blottingTotal RNA was extracted using guanidinium followed by repeated phenoland chloroform:isoamyl alcohol extractions and precipitation. RNA sampleswere separated on 1 % agarose/formaldehyde gel, transferred to reinforcednitrocellulose membranes by capillarity and prehybridized in hybridizationbuffer [50% formamide, 3.6 x SSPE, 0.1% SDS, 10% dextran sulfate,5 x Denhardt's (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% BSA),0.1 mg/ml sonicated herring sperm DNA] at 42°C for 4 h. A random primer-labeled double-stranded DNA probe encompassing nt 8523 -9113(previously described as probe C; Verdin, 1991) was denatured and incubatedwith the membrane at 42°C for a minimum of 12 h. The membrane waswashed at room temperature twice in 2 x SSPE/0.1% SDS for 15 min eachand twice in 0.2 x SSPE/0.1% SDS for 15 min each and exposed toautoradiographic film at -70°C for 1-4 days.

In vivo and in vitro methylation of DNAExponentially growing ACH2 and Ul cells were pelleted by centrifugation(500 g, 10 min), resuspended at 2 x 107 cells/ml in culture medium anddimethylsulfate was added at room temperature at a final concentration of0.1% for 1-10 min. The reaction was stopped by diluting the cells withcold PBS, centrifugation, followed by another wash. Cells were then lysedin proteinase K buffer and digested as indicated above. DNA was purifiedby repeated phenol/chloroform extractions, digested with BamHI to reduceits viscosity, re-extracted with phenol/chloroform and ethanol precipitated.After resuspending the DNA in water (1 mg/ml), piperidine (finalconcentration 1 M) was added for 30 min at 90°C. Samples were evaporated,resuspended in 1/10 volume of water, evaporated again and resuspendedin water. Control DNA from Ul or ACH2 cells purified as described abovewas treated with DMS or hydrazine in vitro as recommended by Saluz andJost (1990).

Ligation-mediated PCRDNA to be used as a substrate in LMPCR should ideally possess 3' and5' phosphate groups. To fulfill this condition, DNA modified in vitro orin vivo with nucleases or chemical agents was submitted to the followingtreatment. (i) DNA treated with DMS + piperidine has both 3' and 5'phosphate extremities and is thus ideally suited for LMPCR without furthermodification (Mueller and Wold, 1989). (ii) Micrococcal nuclease digestionofDNA results in a 5'-OH group and a 3' phosphate group. Consequently,the 5'-OH extremity was phosphorylated with T4 kinase prior to LMPCR(Pfeifer and Riggs, 1991). (iii) DNase I digestion generates a 5' phosphateextremity and a 3' hydroxyl group. The latter group must be blocked priorto LMPCR since it can serve as a non-specific primer during both the primerextension reaction and PCR (Pfeifer and Riggs, 1991). Blocking of theseextremities was performed with terminal transferase and dideoxy-NTP aspreviously described (Pfeifer and Riggs, 1991).

Ligation-mediated PCR was carried out essentially as described previously(Mueller and Wold, 1989), except that a new linker/primer was used forthe ligation reaction. This linker/primer was obtained after annealing primer

3258

LP (5'-GTATCGATCTGGAGATCTGAATTC-3') with primer LP2(5'-GAATTCAGATC-3'). To examine the 5' LTR only, without interferencefrom the 3' LTR, a set of three primers localized downstream of the 5'LTR was used to examine the upper strand: primer A (nt 774-5'-CTCC-GCTAGTCAAAATTTTTGG-3') was used for the first extension reaction,primer B (nt 760-5'-ATTTTTGGCGTACTCACCAGTC-3') was used inPCR amplification with LP1 and primer C (nt 750-5'-TACTCACC-AGTCGCCGCCCCTCGCCTCTTG-3') was radiolabeled with T4polynucleotide kinase and ['y-32P]ATP, and used in the labeling reaction.

Gel scanning and quantificationSeveral exposures of each gel were scanned using a Molecular Dynamicsgel scanner. Results are presented as arbitrary OD units.

AcknowledgementsThe authors thank Monique Dubois-Dalcq for support, encouragement andsuggestions during the course of this work. We alsothank Heinz Amheiter,Monique Dubois-Dalcq, Lynn Hudson, Malcolm John, Charles Vinson andCarl Wu for their helpful comments and suggestions on this manuscript.We are grateful to Jeffrey Hayes for help with the densitometric analysisand its quantification. We are grateful to Anthony Fauci, Tom Folks andGuido Poli for making their celllines available to us for these studies throughthe AIDS Research and Reference Reagent Program, Division of AIDS,NLAID, NIH. Carine Van Lint is supported by the Belgian-AmericanEducational Fundation (BAEF) and the 'Fonds National de la RechercheScientifique' (FNRS, Belgium).

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Received on March 26, 1993; revised on May 10, 1993

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immunodeficiency virus type 1 during transcriptional

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