Nucleic Acids Research, Vol. 20, No. 2 273-278

The transcriptionally-active MMTV promoter is depleted of histone H1 Emery H.Bresnick, Michael Bustin1, Veronique Marsaud2, Helene Richard-Foy2 and Gordon L.Hager* Laboratory of Molecular Virology and 1Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA and 2Unite de Recherches sur les Communications Hormonales, INSERM U-33, Batiment INSERM, Hopital du Kremlin Bicetre, 80 rue du General Leclerc, 94276 Bicetre Cedex, France Received September 30, 1991; Revised and Accepted December 9, 1991

ABSTRACT We have used an ultraviolet light cross-linking and immunoadsorption assay to demonstrate that histones Hi and H2B are bound to the repressed MMTV promoter. Hormone activation results in reduced Hi content with little or no change in H2B. High resolution analysis of the glucocorticoid-inducible DNasel hypersensitive region demonstrates an NF-1 footprint as well as specific sites of enhanced cleavage on nucleosome B and in the nucleosome B/nucleosome A linker. These results are consistent with a model in which binding of the glucocorticoid receptor to glucocorticoid regulatory elements on the surface of nucleosome B induces a chromatin transition that is necessary for transcription factor (NF-1 and TFIID) recruitment to the MMTV promoter. We hypothesize that association of histone HI with important ciselements on the promoter masks these sites, and glucocorticoid-induced displacement of Hi is necessary to expose factor binding sites at the 3' edge of nucleosome B, in the nucleosome B/nucleosome A linker and at the 5' edge of nucleosome A. INTRODUCTION The agonist-bound GR regulates the expression of a variety of genes by binding to GREs [GGTACANNNTGT(T/C)CT] that are usually positioned upstream of the transcription initiation site (1,2). In vitro transcription studies have provided evidence that steroid receptors regulate transcription from naked DNA templates by facilitating the formation of a preinitiation complex (3-5). We have used the steroid-inducible MMTV promoter as a model regulatory element to study the mechanism of GRdependent transcription initiation (6). Functional analysis of the MMTV promoter has revealed multiple cis-acting elements that are necessary for hormoneinduced transcription. The distal (-186 to -171) and proximal GREs (-115 to -110, -99 to -94, and -84 to -79) are *

To whom

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necessary for hormone induction (7,8), as well as an NF-I binding site (-82 to -56) that overlaps the proximal GRE (9-12) and three binding sites (-60 to -39) for OCT-I (13,14). Richard-Foy and Hager (15) used indirect end-labeling to provide evidence that the LTR regulatory sequences are organized into an array of six positioned nucleosomes (designated A-F), with the GREs positioned on the surface of nuc-B. Activation of the promoter by glucocorticoid hormone results in a HSR from approximately -60 to -250, a region that encompasses nuc-B. Cordingley et al. (16) used in vivo ExoIllI footprinting to show that NF-I and TFIID are recruited to their cognate binding sites on the MMTV promoter in a hormonedependent manner. The apparent concentrations and DNAbinding activities of these factors are unaffected by hormone (17). Based on these observations, it was proposed that the specific positioning of nuc-B might occlude the NF-1 site, and the hormone-induced HSR reflects a chromatin transition that is necessary to expose the NF-1 site (15,18). Histone HI interacts with linker regions of chromatin (19). In some cases, active chromatin has been shown to be partially depleted of HI (20). As HI is involved in compacting polynucleosomal arrays into higher order structures (19,21), depletion of HI may have functional consequences for both transcription initiation and elongation. For example, the depletion of HI from a nucleosomal template could increase the efficiency of elongation by RNA polymerase. In at least one clear example, however, HI has been directly observed on an actively transcribed Balbiani ring gene by an electron microscopy technique (22). HI depletion could also affect initiation by unmasking cis-acting elements and, therefore, increasing the accessibility of these elements to their respective transacting factors. The specifically organized MMTV chromatin template provides a unique system to address a possible regulatory role for HI in transcription. As transcription factor binding sites are present at the 3' edge of nuc-B (NF-1), in the nuc-B/nuc-A linker (OCT-1), and at the 5' edge of nuc-A (TFIID) (15,23,26), we have

274 Nucleic Acids Research, Vol. 20, No. 2 asked if HI is associated with nucleosomes on the MMTV promoter. We demonstrate that histones H 1 and H2B are bound to the repressed MMTV promoter, and hormone activation decreases the HI content of the LTR. In addition, we use a primer extension assay (24) to ask what is the molecular change that occurs in the nuc-B region that causes nuclease hypersensitivity? We show that the low resolution DNaseI HSR results from enhanced cleavage of bonds on nuc-B and throughout the nucB/nuc-A linker. The implications of these results are discussed with respect to the mechanism of transcription factor recruitment.

MATERIALS AND METHODS Cell culture and plasmids Cells (904.131) were obtained by transfection of the chimeric

appropriate histone in chromatin and nucleosomes was also demonstrated (28,29). Supernatants were incubated with polyclonal antisera (8 ,tl) for 1 h at 4°C, followed by protein-ASepharose CL-4B (20 1l pellet) for 2 h at 4°C with constant rotation. Sepharose pellets were washed 4 times with 1 ml aliquots of 10 mM TES (pH 7.6), 4 mM EDTA, 10% glycerol and 50 mM NaCl. Each wash was performed by vortexing for 20 s, followed by centrifugation for 30 s at 15600 x g. Washed pellets were incubated in 10 mM Tris-HCl (pH 7.6), 0.1 mM EDTA (0.2 ml), containing 0.5 mg/ml proteinase K for at least S h at 37°C. Immunoadsorbed DNA fragments were purified by adding 20 izg of tRNA and extracting twice with phenol/chloroform, followed by once with chloroform and ethanol precipitation.

BPV-based construct pM 18 into C 127 cells and grown as described previously (16,25). Plasmid pM18 contains the MMTV LTR driving the v-Ha-ras gene and is present at approximately 200 copies that are integrated into chromosomal DNA as tandem repeats.

Nuclei isolation and nuclease digestions Nuclei were isolated from 2 x 108 cells that had been treated for 1 h with 100 nM dex or ethanol vehicle as described previously (26). DNaseI digestions and purification of genomic DNA were as described previously (15). For in situ restriction enzyme digestions, washed nuclei were resuspended in 0.2 ml of nuclei wash buffer (to a final A260 of 40) containing 10 mM MgCl2 and 150-750 units/ml of MboI (Pharmacia) and were incubated for 30 min at 30°C. The reactions were terminated, and genomic DNA was purified as described previously (26). UV cross-linking Nuclei from 904.131 cells (2.5 x 108) were resuspended in 2 ml of nuclei wash buffer (to a final A260 of 60-80) in 35 mm x 10 mm tissue culture dishes (Coming) at 4°C. PMSF (0.1 mM) and leupeptin (10 jg/ml) were included in all buffers. Nuclei suspensions were placed 10 cm below an inverted transilluminator (Ultra-violet Products Inc.) with the filter removed and irradiated for 10 min at 4°C. After transfer to 1.5 ml microfuge tubes, nuclei were collected by centrifugation for 30 s at 7800 x g and were resuspended in 1 ml of 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10 Itg/ml leupeptin, 0.1 mM PMSF. Nuclei were divided into 5, 0.20 ml aliquots, and each aliquot was incubated with 150 units of HaeIII (NEB, 10 ulAI) for 45 min at 30°C. The reactions were terminated with 0.20 ml of nuclei wash buffer containing 25 mM EDTA. Chromatin was solubilized by rotating samples for S min at 4°C, followed by centrifugation for 5 min at 15600 x g. In a representative experiment, 7% of the total A260 units and 9% of MMTV sequences (detected by Southern blot analysis) were solubilized with HaeIHI. Glucocorticoid treatment did not affect the amount of MMTV chromatin solubilized. SDS-PAGE analysis of proteins in extracts from untreated and dex-treated cells revealed identical amounts of H 1 and core histones (data not shown).

Immunofractionation Antisera were elicited in rabbits with purified histone fractions, and the sera were characterized by Elisa, immunoblotting and radioimmunoassay (27). The reactivity of sera with the

Figure 1. Glucocorticoid-induced sensitivity of the MMTV promoter to MboI cleavage. Panel A-Southern blot analysis. Nuclei were isolated from untreated and dex-treated 904.131 cells and digested with 0, 30, 60, or 150 units of MboI. Genomic DNA was purified, digested to completion with PstI and analyzed by Southern blot analysis with a v-ras probe. Fragments that result from cleavage in the nuc-C/nuc-D linker, nuc-B, 30S and v-ras are indicated by arrows. Treatment protocols, positions of the MboI sites, and the position of the probe are diagrammned at the top. Panel B -Quantitative analysis. Bands were excised from the blot in panel A, and bound radioactivity was quantitated by scintillation counting. Background values were obtained by excising regions of lanes 1 and 2 adjacent to the appropriate band in lanes 3 - 7. These values ( 170 and 209 cpm for nuc-B and C-D linker, respectively) were subtracted from the cpm for each band to yield specific values. Specific values were normalized to the sum of all fragments, including parental, in each lane; open bar, nuc-B cleavage, vehicle; solid bar, nuc-B cleavage, dex; diagonal line bar, C-D linker cleavage, vehicle; vertical line bar, C-D linker cleavage, dex.

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Nucleic Acids Research, Vol. 20, No. 2 275

Oligonucleotide primers Oligonucleotides were synthesized on an Applied Biosystems 380B synthesizer. Primers are referred to by number and correspond to the following positions relative to the MMTV RNA initiation site: #343 (-105 to -129), 5'-AGCTCAGATCAGAACCTTTGATACC-3' #344 (-200 to -176), 5'-TTAAGTAAGTTTTTGGTTACAAACT-3' #397 (+65 to +39), 5'-GTGACGAGCGGAGACGGGATGGCGAAC-3'

Primer extension analysis of genomic DNA and immunoadsorbed DNA fragments Gel-purified oligonucleotide primer (5 pmoles) was labeled with [32P]ATP and polynucleotide kinase. Genomic DNA (10 ug) or 25% of total immunoadsorbed DNA fragments were incubated in 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 3 mM MgCl2, 0.05% nonidet-P40, 0.05% Tween-20, 0.8 mM dATP, dCTP, dGTP, dTTP (or appropriate dideoxy NTP mixes), 0.4 pmoles of end-labeled primer (6-20 x 106 cpm), and 2.5 units of Amplitaq (Perkin Elmer Cetus) in a final volume of 30 y1. Thirty cycles of primer extension were carried out using an annealing temperature of 2°C below the oligonucleotide melting temperature, calculated from its%GC content. Samples were extracted once with phenol/chloroform, ethanol precipitated, and analyzed on 8% acrylamide-urea sequencing gels. Haelil

RESULTS

Hormone responsiveness of MMTV templates in 904.131 cells To ask if HI is bound to MMTV regulatory sequences we employed the 904.131 cell line, which contains approximately 200 copies of MMTV-ras fusions that are stably integrated into chromosomal DNA. As our goal was to compare the HI content of repressed and transcriptionally active templates, it was necessary to determine the percentage of honnone-responsive templates. MboI, SstI and DdeI cleave sites on the 3' half of nucB in isolated nuclei much better after hormone treatment (26,30,3 1). Fig. IA shows an experiment to measure the percentage of MMTV templates in 904.131 cells that develop hypersensitivity to MboI in the nuc-B region. Nuclei from untreated or dex-treated cells were digested with increasing amounts of MboI. Genomic DNA was purified, digested to completion with PstI, and MboI cleavage products were detected by Southern blot analysis with a v-ras probe. Bands were excised from the blot, and the amount of specifically-bound probe was measured by scintillation counting. The ratio of nuc-B fragment to total fragments increases in a hormone-dependent manner to 0.22 at 150 units of MboI (Fig. IB). In contrast, the ratio of nuc-C/nuc-D linker fragment to the parental fragment remains constant. and cleavage at additional sites in v-ras and 30S is also unaffected. This experiment demonstrates that a minimum of 18%

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Figure 2. Direct evidence that histone HI is bound to the repressed MMTV A-Immunoadsorption with histone antisera. Chromatin from untreated or irradiated nuclei was solubilized with HaeIII, and cross-linked DNA fragments were immunoadsorbed with nonimmune serum (NI) or antisera against histones HI, H2B or H3. DNA fragments were purified and analyzed by primer extension (343 primer). Treatment protocols and positions of the HaeUl sites are diagrammed at the top. Bands that result from HaeI cleavage at the -223 site are indicated with an open arrow. Lane 1, control extract from nuclei that were not digested with HaellI; Lanes 8 and 9, dideoxy A and C markers with plasmid pLTRluc template, respectively. Panel B-Irradiation requirement for immunoadsorption of MMTV fragments. Chromatin from irradiated and untreated nuclei was solubiized, and DNA fragments were immunoadsorbed with nonimmune or anti-HI serum. Treatment protocols are diagrammed at the top. promoter in 904.131 cells. Panel

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Figure 3. Glucocorticoid-induced decrease in the amount of promoter-associated histone HI. Nuclei from untreated and dex-treated 904.131 cells were irradiated, chromatin was solubilized, and DNA fragments were immunoadsorbed with antisera against histones HI or H2B. DNA fragments were purified and analyzed by primer extension (343 primer). Treatment protocols and positions of the HaeIlI sites are diagrammed at the top. Bands that result from HaeIII cleavage at the -223 and -636 sites are indicated by open and solid arrows, respectively. Lane 1, control extract from nuclei that were not digested with HaeIII; lane 2, control extract from uninduced nuclei that were digested with HaeIII but not immunoadsorbed; lane 3, control extract from induced nuclei that were digested with HaeUI but not immunoadsorbed; lanes 4-9, extracts from hormone-treated or untreated nuclei that were digested with HaeIII and immunoadsorbed as indicated; lane 10, dideoxy C marker with plasmid pM5O template.

276 Nucleic Acids Research, Vol. 20, No. 2 of the templates develop hypersensitivity to Mbol in a hormonedependent manner [0.22 (+dex)-0.04 (-dex)x 100].

Direct evidence that histone HI is bound to the MMTV promoter We used a UV cross-linking and immunoadsorption assay to ask if HI is associated with LTR nucleosomes in 904.131 cells. Nuclei were isolated from dex or vehicle-treated cells and were irradiated with UV light as described by Kamakaka and Thomas (20). Chromatin was solubilized by HaeIII digestion, and fragments were immunoadsorbed with either specific or nonspecific antisera. Specific fragments were detected by Taq polymerase primer extension analysis of the deproteinized, immunoadsorbed DNA with an end-labeled primer specific for the MMTV promoter. As shown in Fig. 2A, a HaeIII fragment of the MMTV promoter that encompasses 4 nucleosomes (nuc-B, nuc-A, nuca and nuc-b) can be specifically immunoadsorbed with antisera directed against histones HI, H2B and H3. The relative efficacy of these antibodies to immunoadsorb the HaeIII fragment (HI > H2B > H3) agrees with earlier studies on the accessibility of histone epitopes in chromatin (28,29). The fragment is not adsorbed by nonimmune serum, and the presence of the appropriate extension product is absolutely dependent upon HaelI cutting. Several other bands that appear in lane 3 represent Taq polymerase termination sites preceding two or more thymine residues, sites of UV-induced thymine-thymine dimers. As only trace amounts of MMTV fragments are detected when chromatin from non-irradiated cells is immunoadsorbed (compare lanes 2 and 4 of Fig. 2B), the presence of the MMTV fragment in the pellet requires the cross-linking of histones to DNA. Since H1 removal may be necessary for NF-1, OCT-1 or TFIID binding, we examined the effect of dex on the association of HI with the promoter. As shown in Fig. 3, identical amounts of the HaeIII fragment are released from dex and vehicle-treated nuclei (compare lanes 2 and 3). The HaeIII fragment is specifically immunoadsorbed with HI antiserum (compare lane 6 with lane 4), and dex treatment results in a 45.5 4 15.8% [(X i SE), n = 8] decrease in HI. The MMTV promoter is maximally-induced under these conditions as shown by nuclear run-on assays2. In contrast to the decreased association of HI with the activated MMTV promoter, the amount of HaeIII fragment immunoadsorbed with H2B antiserum is only slightly affected by dex (compare lanes 8 and 9) [% decrease = 10.7 ± 2.0 (X i SE), n = 6]. An additional band at the top of the gel in Fig. 3 results from annealing of the 343 primer to a small population of fragments that were not cut at the -223 HaeIII site but were cut at a HaeIII site (-636) in the nuc-D/nuc-E linker. Incomplete cleavage at the -223 site is due to inefficient restriction enzyme cleavage of chromatin. The amount of HI bound to this fragment decreases to a similar degree as the tetranucleosomal fragment, whereas the amount of H2B was unaffected by hormone treatment. The HaeILH tetranucleosome fragment can be subdivided, albeit inefficiently, into 2 dinucleosome fragments by digestion with HaeIII and BamH 1 simultaneously to yield fragments encompassing nuc-B/nuc-A and nuc-a/nuc-b. The BamHI site at + 107 is within the 3' portion of nuc-A, and, therefore, the yield of dinucleosome fragment is low. A comparable decrease in association of HI with the nuc-B/nuc-A fragment was observed; equivalent amounts of this fragment were adsorbed with antiH2B antiserum (data not shown). These results demonstrate that

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Figure 4. High resolution analysis of the glucocorticoid-inducible DNasel hypersensitive site on the MMTV promoter. Panel A-Primer extension analysis of the HSR. Nuclei were isolated from untreated or dex-treated 904. 131 cells and digested with 10 u/ml of DNasel. Genomic DNA (chromatin) and DNaseldigested plasm-id DNA (free) were analyzed by primer extension (397 primer). Symbols: open circles, bands that are repressed by dex;, closed circles, bands that are enhanced by dex. Panel B-Densitometric profile of DNasel cleavage pattern of transcriptionally inactive (solid line) and active MMTV chromatin (dotted line). The apparent hormone-dependent hypersensitivity at - 128 is a gel artifact.

substantial H 1 depletion occurs without a comparable decrease in a core histone. To further investigate the hormone-induced chromatin transition, we used a high resolution assay to examine the DNaseI HSR on the MMTV promoter.

Evidence that the glucocorticoid-inducible DNasel on the MMTV promoter is centered on the nuc-B core and the nuc-B/nuc-A linker DNasel HSRs have been attributed to nucleosome-free gaps in an otherwise ordered chromatin structure (32,33). Here, we utilize a Taq polymerase primer extension assay to examine the HSR on the MMTV promoter in high resolution. Analysis of genomic DNA from 904.131 cells, which have a strong hormonedependent DNaseI HSR on agarose gels (data not shown), reveals a series of bands that are enhanced (closed circles) or repressed (open circles) in response to dex (Fig. 4A). Many of the hormone-induced alterations in DNasel cleavage fall on or near factor binding sites, i.e. TETID (16,17), OCT-i (13,14), MP3 (34), and GR (35,36). In addition to single bp sites of altered

hypersensitive site

Nucleic Acids Research, Vol. 20, No. 2 277

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Figure 5. Model of the specific chromatin organization of the MMTV promoter. Panel A-Nucleosome boundaries inferred from low resolution (15,26) and high resolution (23) MNase mapping data were used to assign positions of dyad symmetry (+). Regions of 146 base-pairs were designated based on the nucleosome dyads and are shown below the schematic. The DNaseI HSR is shown above the schematic: open bar, cuts on coding and noncoding strands; hatched bar, singlestranded cuts on the coding strand. Transcription factor binding sites are indicated by hatched boxes: GRE-d, distal GRE; GRE-p, proximal GREs; NFI, nuclear factor 1; OCTI, octamer binding protein 1; TFIID, transcription factor IID; Afl2, Mbol and Sstl sites are hypersensitive in the hormone-activated promoter. Panel B-Occlusion of the MMTV promoter by histone HI; B, nuc-B; A, nuc-A.

sensitivity, a footprint (bordered by hypersensitive cuts) is observed from -54 to -81. This footprint further documents hormone-dependent occupancy of the NF-i site (16). The lack of continuous TFIID and OCT-I footprints may reflect disruption of the multiprotein preinitiation complex during DNaseI digestion, as these factors have inherently lower affinity for their binding sites compared to NF-1. Densitometric analysis (Fig. 4B) of samples from vehicle (solid line) and dex-treated cells (dotted line) reveals that a subset of bonds become more accessible to DNaseI. These bonds are clustered on nuc-B (predominantly the 3' half, -125 to -75), throughout the nuc-B/nuc-A linker (-75 to -23), and at the 5' end of nuc-A (-23 to -5). Hormone-induced hypersensitivity, therefore, is not restricted to the nuc-B core (Fig. SA).

DISCUSSION Association of histone Hi and

core

histones with MMTV

regulatory sequences The experiment in Fig. 1 demonstrates that approximately 20% of the MMTV templates in 904.131 cells develop MboI hypersensitivity in a hormone-dependent manner. This represents a minimal estimate, beause extensive digestion of nuclei leads to an overall degradation of nuclear structure, precluding a limit digest. As a significant amount of template must be occupied by a sequence-specific DNA binding protein to visualize a footprint, the clear NF-I footprint in Fig. 4A provides further evidence that a significant portion of the MMTV fusions are hormone-

responsive. The amount of HI cross-linked to the LTR decreases by 46%, in approximate agreement with the measurements of hormone-responsive templates. Our experiments provide the first example of Hi bound to a well-defined nucleosomal array and decreased HI content in response to a single regulatory molecule, the hormone-activated glucocorticoid receptor. Although significantly less HI is cross-linked to the active promoter, we can not distinguish between the following two mechanisms that would lead to decreased cross-linking; (i) complete dissociation of HI from the nucleoprotein template, and (ii) an altered conformation of promoter-bound HI that reduces the crosslinking efficiency. At high resolution (Fig. 4), the DNaseI HSR is not positioned symmetrically on the nuc-B core, but is displaced to the 3' side. The limitation of hypersensitive bonds to the coding strand downstream of position -50 provides an explanation for the discrepancy between the position of the DNaseI HSR determined by agarose gels (15) and that reported here. Single-stranded cuts would not be detected in the agarose gel conditions used in lowresolution studies, but are detected in denaturing gels. The assymetry suggests that the chromatin transition on the promoter that causes the DNaseI HSR does not necessarily result from nucB removal. We argue that the DNaseI hypersensitivity is a composite profile that results from (i) increased nucleolytic attack at specific sites due to factor binding, and (ii) a regional increase in sensitivity due to a B nucleosome structural transition. The exact nature of this transition remains to be determined. Resistance to MNase cleavage in the nuc-A and nuc-B core regions in chromatin from induced cells (15,23,26) is also consistent with the continued presence of octamer cores. Taken together with the H2B cross-linking results, these observations are consistent with retention of either native or modified (37) nucleosomes on the LTR during transcription. Other studies have also suggested that core histones remain associated with coding regions (38,39) and regulatory regions (40) of transcribed genes.

Mechanism of NF-1 recruitment As the proximal GRE and the NF-I consensus element partially overlap (Fig. SA), and the GR inhibits NF-l binding to its recognition site in vitro (12), it is unlikely that recruitment of NF-1 results from cooperative protein-protein interactions between the GR and NF-1. In addition, NF-I can activate transcription efficiently in vitro from naked DNA templates in the absence of GR (41, M.G. Cordingley and G.L. Hager, unpublished data). These arguments support a role for a specific nucleoprotein structure in preventing constitutive activation of the MMTV promoter by basal transcription factors. The following evidence led to the hypothesis that GR-induced alteration of nuc-B structure may be necessary for NF- 1 loading. (i) The hormone-induced HSR correlated with the position of nuc-B (15). (ii) NF-l is excluded from its recognition site in vivo in uninduced cells (16,42). (iii) The GR can bind to reconstituted nuc-B monosomes (43,44) and nuc-B/nuc-A disomes in vitro (31), whereas NF-l is unable to bind (31,44), despite high affinity for its binding site in naked DNA [KD = 10-12 (45)]. We recently demonstrated by in vivo footprinting that NF-l binds constitutively to transiently transfected MMTV DNA, whereas it is excluded from stable, replicating templates in the same nucleus, providing direct evidence for the chromatin exclusion model (50). The in vitro experiments suggest a model of NF- 1 exclusion from a nucleosomal template based on the interaction of NF- 1

278 Nucleic Acids Research, Vol. 20, No. 2 with the core surface. Several mechanisms of exclusion could be suggested, including rotational orientation of recognition sites on the core surface, or steric blockage resulting from DNA positioned in the histone environment. The interpretation of these experiments is complicated by the fact that reconstituted molecules can exist in multiple translational frames (26), and, in one case, the NF-1 site is well within the reconstituted core (44), differing from the in vivo position (15,23,26) at the 3' edge of nuc-B. The pronounced increase in sensitivity to cleavage by restriction enzymes whose sites lie on the surface of nuc-B (summarized in Fig. 5A) indicates that some alteration is occurring in nuc-B. If core histones remain associated with the template during transcription, what could be the molecular basis for the chromatin transition that allows recruitment of NF-I? One mechanism could involve an altered interaction of histone HI with nuc-B, and a destablized nuc-B structure.

Is Hi depletion necessary for activation? Histone HI can dramatically influence the transcriptional efficacy of specific templates in vitro (46-48), and it has been reported that HI interacts with the NF-1 consensus element (49). The NF-I site on the MMTV promoter is in the nuc-B/nuc-A linker at the 3' edge of nuc-B, the OCT-I sites are in the nuc-B/nuc-A linker and the TATA box is at the 5' edge of nuc-A. As histone HI would be expected to interact with this region (19), one can envision a model in which the promoter is repressed by an HI-stabilized nucleoprotein structure. Formation of a large complex consisting of one to four homodimers of GR (95,000 Da/monomer) with nuc-B could directly expose these sites by disrupting the binding of HI to the linker (Fig. 5B), as well as altering the core structure itself. Alternatively, localized disruption of HI-chromatin complexes could indirectly expose binding sites by impairing the ability of HI to compact nucleosomal arrays into a 30 nM chromatin fiber (21). In conclusion, we have demonstrated that HI is bound to a fragment of the MMTV promoter that contains important cisacting elements, and we propose that depletion of HI from the LTR is an initial response to complex formation between the GR and nuc-B and is necessary for preinitiation complex assembly. It may be possible to further refine the UV cross-linking and immunoadsorption assay to examine the association of HI and core histones with individual nucleosomes that are positioned on nontranscribed regulatory regions or on transcribed sequences. The availability of antibodies that recognize HI subtypes and modified (e.g., acetylated and phosphorylated) histones will allow one to compare the relative distribution of these minor histone species in individual nucleosomes.

ACKNOWLEDGEMENTS We thank Ronald G. Wolford for oligonucleotide synthesis, Charles Rories for supplying purified oligonucleotides, Diana S. Berard for assistance with cell culture, and Mitsuhiro Shimizu for introducing us to the Taq primer extension assay. We thank Philippe LeFebvre, Sam John and Alan Wolffe for helpful comments on the manuscript.

4.

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26. 27. 28. 29. 30.

31. 32. 33. 34. 35.

36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

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