From INSERM U459, Laboratoire de Biochimie Structurale, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille cedex, France
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ABSTRACT |
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The retinoic acid signaling pathway is controlled
essentially through two types of nuclear receptors, RARs and RXRs.
Ligand dependent activation or repression of retinoid-regulated genes is dependent on the binding of retinoic acid receptor
(RAR)/9-cis-retinoic acid receptor (RXR) heterodimers to retinoic acid
response element (RARE). Although unliganded RXR/RAR heterodimers bind
constitutively to DNA in vitro, a clear in vivo
ligand-dependent occupancy of the RARE present in the
RAR2 gene promoter has been reported (Dey, A., Minucci, S., and
Ozato, K. (1994) Mol. Cell. Biol. 14, 8191-8201).
Nucleosomes are viewed as general repressors of the transcriptional
machinery, in part by preventing the access of transcription factors to
DNA. The ability of hRXR
/hRAR
heterodimers to bind to a
nucleosomal template in vitro has therefore been examined.
The assembly of a fragment from the RAR
2 gene promoter, which
contains a canonical DR5 RARE, into a nucleosome core prevented hRXR
/hRAR
binding to this DNA, in conditions where a strong interaction is observed with a linear DNA template. However, histone tails removal by limited proteolysis and histone hyperacetylation yielded nucleosomal RAREs able to bind to hRXR
/hRAR
heterodimers. These data establish therefore the role of histones NH2
termini as a major impediment to retinoid receptors access to DNA, and identify histone hyperacetylation as a potential physiological regulator of retinoid-induced transcription.
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INTRODUCTION |
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Core histones H2A, H2B, H3, and H4 are the main protein components of chromatin around which DNA is wrapped in 146-bp1 segments, forming stable nucleosomal structures. Nucleosome spacing and histones post-translational modifications, and most notably acetylation, varies greatly from one chromosomal domain to another (with hyperacetylated histones being preferentially associated to transcriptionally active chromatin) and have strong effects on gene activity (reviewed in Refs. 2 and 3). Beside these long range effects, chromatin assembly on regulatory regions of eukaryotic promoters affects directly the transcriptional activity of genes. Due to the organization of these DNA sequences into precisely positioned arrays of nucleosomes, transcription factors access to their cognate response elements is in most cases restricted and nucleosome assembly is therefore viewed as a general cellular strategy to repress transcription. In vitro assembly of nucleosomes on short DNA fragments documented this type of effect for many DNA-binding proteins, including Gal4 (4), SP1 (5), nuclear factor 1 (6), heat shock factor (7), and TATA-binding protein (8), and genetic experiments in yeast using the PHO5 transcription unit have established a link between transcriptional activation and chromatin structure disruption (reviewed in Ref. 9). However, chromatin organization is also, in some instances, a mean to favor transcriptional activity of genes, as described for the estrogen-regulated vitellogenin B1 gene (10). Therefore, transcription regulation must be viewed as a complex set of interactions involving specific transacting factors, general transcription factors, and coactivators recruitment onto a nucleoprotein complex. The productive interaction of DNA-binding proteins with their cognate response elements is thus conditioned by two parameters: (i) structural features of the nucleosome and (ii) dynamic processes leading to the alteration of chromatin structure by macromolecular complexes such as SWI/SNF (4, 11), which is associated to RNA polymerase II under low stringency conditions (12), nucleosome remodeling factor (13), or nucleoplasmin and NAP-1 (14). Structural features of chromatin also implies a competition between chromatin constituents and transcriptional coactivators (some of which bearing strong structural similarities with histones H3 and H4 (15)), which will eventually determine the overall transcriptional activity of genes (reviewed in Ref. 16).
In an effort to better understand the role of chromatin structure in
nuclear receptors binding to their response elements, we have used
purified components to investigate the impact of nucleosome assembly on
a promoter containing a prototypical retinoic acid response element
(RARE). Retinoic acid receptors (RARs and RXRs) heterodimers bind,
in vitro, to RAREs with high affinity, irrespective of the
presence of ligand. On the contrary, Ozato and co-workers (1)
established, by in vivo footprinting experiments, that
agonist treatment of target cells is an absolute prerequisite for
heterodimers binding to the RARE of the RAR gene, as it is to
observe biological effects of retinoids in vivo. This
agonist-dependent occupancy of hormone response element was
also observed with the glucocorticoid receptor (17). The
transcriptional and DNA binding activities of these nuclear receptors
are therefore controlled in vivo at multiple levels, which
include post-translational modifications (see for examples, Ref. 18 and
19) and by epigenetic mechanisms (reviewed in Refs. 20 and 21).
The transcriptional activation observed in the presence of RAR and RXR is triggered by binding of heterodimers to RAREs that consist, in most cases, of a direct repeat (DR) of the sequence PuGGTCA. A direct repeat of the hexanucleotide PuG(G/T)TCA spaced by five nucleotides favors the binding of RXR/RAR heterodimers, whereas a spacing of four, three, or one base converts the direct repeat into a thyroid hormone, vitamin D, and 9-cis-RA or PPAR response element, respectively (Ref. 22, and reviewed in Ref. 23). RXR/RAR heterodimers display the highest affinity for half-sites spaced by 5 bp (DR5) and a lower affinity for half-sites having a 2-bp spacing. DR1 RAREs accommodate heterodimer binding in an opposite polarity (i.e. RAR being the 3'-bound receptor) (reviewed in Ref. 24). Nucleosome assembly on DNA sequences containing such direct repeats should therefore introduce two new types of constraints upon RXR/RAR heterodimer binding to DNA, as a consequence of the helical nature of the DNA. The first type of constraint is defined by the translational phasing of the nucleosome, which describes 5' and 3' boundaries of the octamer core on the linear DNA sequence and thus identify the dyad axis of the nucleosomal core particle. Transcription factors access to their cognate DNA-binding site is facilitated when protein-DNA interactions take place close to nucleosome boundaries, reflecting a looser interaction of DNA with histones, as demonstrated for the glucocorticoid receptor (25). The second type of constraint is the rotational phasing within the nucleosome core, which reflects the orientation of any segment of DNA relative to the core histone surface (26).
In this work, we have first examined the positioning of histone
octamers in vitro on a retinoid-regulated promoter, the P2 promoter of the human retinoic acid receptor (RAR-
2) that
contains a DR5 response element (
-RARE (27)). We find that
nucleosomes assembled spontaneously on DNA fragments from the RAR
gene P2 promoter at a position placing the
-RARE at the dyad axis of the core particle. Binding of purified hRAR
/hRXR
heterodimers was
prevented by the wrapping of the RARE around the native octamer. On the
contrary, heterodimer binding could be observed when histone tails were
removed by limited proteolysis and when histones were hyperacetylated,
suggesting a critical role of this post-translational modification in
the regulation of the DNA binding activity of retinoids receptors
in vivo.
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EXPERIMENTAL PROCEDURES |
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Materials
Antiproteases, trypsin, and trypsin inhibitor were purchased
from Sigma. Taq DNA polymerase was from Life Technologies,
Inc. (Rockville, MD); isopropylthio--galactopyranoside, ampicillin, and kanamycin were from Appligene/Oncor (Strasbourg, France). Restriction enzymes and agarose were from Promega (Madison, WI), oligonucleotides were purchased from Eurogentec (Le Sart-Tilman, Belgium). Acrylamide and bisacrylamide mixture (Protogel) were from
National Diagnostics (Atlanta, GA).
Bacterial and Eukaryotic Cell Lines
The JM109 bacterial strain was used for all subcloning
procedures, whereas the M15 strain (Qiagen) was used for the
overexpression of both His6-hRAR and
His6-Flag-hRXR
(18). HeLa cells were used as a source
for core histones and grown in Dulbecco's minimal essential medium
containing 10% fetal calf serum, penicillin, and streptomycin (100 units/ml) to 80-90% confluency prior to harvesting and histone
octamer extraction.
Core Histone Purification
Core histones were prepared essentially as described in
Côté et al. (32). The entire procedure was
carried out at 4 °C. HeLa cells from 20 T225 flasks were collected
in 1 × phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.4 mM KH2PO4, pH 7.4) and washed
twice with this buffer. 20 pellet volumes of buffer CLB (20 mM Tris-HCl, pH 8.0, 3 mM MgCl2,
0.25 M sucrose, 0.5 mM phenylmethylsulfonyl
fluoride) with 0.5% Nonidet P-40 were added and cells broken by 10-15
strokes in a Dounce homogenizer. Lysate was centrifuged for 20 min at
4,000 × g and the nuclei pellet washed twice with
buffer CLB. 50 ml of buffer TNE400 (10 mM
Tris-HCl, pH 8.0, 0.4 M NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 5 µM
leupeptin) was added for 12 mg of DNA and the mixture was stirred
gently for 15 min. Nuclei were pelleted and washed once with buffer
TNE400, then resuspended in buffer PB600 (50 mM NaPO4, pH 6.8, 0.6 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 5 µM
leupeptin). While stirring, 20 g of dry hydroxyapatite (Bio-Gel HTP, Bio-Rad) was added per 12 mg of DNA. The slurry was poured in a
Econo column (5.0 × 20.0 cm, Bio-Rad) and the flow-through was
discarded. The resin was washed with 15 volumes of buffer PB600 and core histones were eluted from the matrix with
buffer PB2500 (50 mM NaPO4, pH 6.8, 2.5 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin). OD280 was
monitored and fractions containing more than 2 mg/ml histones were
pooled and stored at 80 °C until use. Hyperacetylated histones
were prepared as above except that cells were treated for 18 h
with 10 mM sodium butyrate (Sigma). Cross-linking
experiments were performed using dimethyl suberimidate (Pierce) to a
final concentration of 1 mg/ml.
Histones were fractionated on 17% polyacrylamide, 0.9 M acetic acid, 6.25 M urea as described by Panyim and Chalkley (29) on 20-cm gels. Gels were run at 20 mA for 15 h and stained with Coomassie Blue. In this system, the expected order of migration is H1-H3-H2A, H2B-H4 (from top to bottom).
Retinoic Acid Receptors Purification
Full-length hRAR and hRXR
were purified by metal chelate
affinity chromatography to homogeneity as described previously (30).
hRAR
was expressed as a NH2-terminal fusion protein with a His6 tag, whereas hRXR
was fused to the
His6 tag upstream of the Flag epitope (IBI-Kodak,
Rochester, NY). hRXR
was eluted from the affinity matrix by
enterokinase cleavage, thereby removing the His6 tag from
the protein. Both receptors were expressed in the M15 Escherichia
coli strain. Monoclonal antibodies directed against
MRGS-His6 and Flag epitopes were purchased from Qiagen and
IBI-Kodak, respectively.
Plasmids and DNA Probes
The bacterial expression vectors pQE9-hRAR and
pQE9-His6-F-hRXR
have been described previously (18).
Sequences containing the wild type
-RARE response element were
isolated from pPro-RAR
(27) by restriction enzyme digestion.
Milligram amounts of plasmid DNA were cut and selectively
dephosphorylated at one end by calf intestine alkaline phosphatase
(Promega), and the resulting insert was purified by agarose gel
electrophoresis and electroelution. After phenol/chloroform extraction,
DNA fragments were ethanol-precipitated and quantified. 10 pmol were
labeled with T4 polynucleotide kinase and purified by gel-filtration
using standard procedures (31).
Reconstitution of Nucleosomes
Nucleosomes were reconstituted essentially as described in Ref.
32, except that the dialysis step was replaced by a serial dilution in
buffer NRB (25 mM Tris-HCl, pH 7.4, 1.2 mM
MgCl2, 5 mM -mercaptoethanol, 5% glycerol)
from 2 M NaCl to 1.5, 1.2, 0.8, 0.6, and 0.3 M
NaCl to reach a final volume of 200 µl. Typically, 50-100 ng of
end-labeled probe (1-5 × 106 cpm, 300 fmol) were
mixed with varying core histones amounts, so as to obtain a histone:DNA
mass ratio ranging from 0.5 to 3, 0.5 µg of salmon sperm DNA, and 0.5 µg of bovine serum albumin. Naked DNA controls were obtained by
performing similar dilutions in the absence of histones, which were
added after the last dilution step. Aliquots of each reconstitute were
analyzed on a 4.5% polyacrylamide gel in 0.5 × TBE (1 × TBE is 90 mM Tris base, 90 mM boric acid, 2 mM EDTA) and samples containing more than 90% of
reconstituted octamer-DNA complexes were used in further experiments.
The final concentration of nucleosome core particles was around 5-10
fmol/µl.
DNase I and Exonuclease III Protection Assays
Reconstitutes or naked DNAs were treated with either DNase I or exonuclease III prior to DNA extraction and resolving of DNA fragments on 6% urea denaturing polyacrylamide gels. 15 µl of reconstitution or control mixtures (about 30,000-40,000 cpm) were brought to 5 mM MgCl2 and CaCl2 and 1 unit of DNase I (Worthington, Freehold, NJ) was added. Digestions were for 0 to 8 min at room temperature (~22 °C), and stopped by addition of 100 µl of stop buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). 10 µg of proteinase K was added per sample and incubated overnight at 37 °C. DNAs were extracted with phenol and phenol/chloroform prior to ethanol precipitation. Exonuclease III protection experiments were run similarly except that samples were brought to 4 mM MgCl2, and digested with 200 units of exonuclease III (Appligene/Oncor, Strasbourg, France).
Linear amplification of the coding strand was performed using standard polymerase chain reaction conditions (33) and the 19-mer oligonucleotide 5'-ACACAGAATGAAAGATTGAATT-3'. 1 pmol of 5'-end labeled primer was used in a reaction mixture containing 1.5 mM MgCl2 and ~30 femtomoles of DNase I-treated, non-labeled DNA. Thermocycling was performed using 25 cycles of 94 °C for 30 s, 41 °C for 1 min, 72 °C for 30 s in a Perkin-Elmer 2400 thermocycler. Amplification products were phenol-chloroform extracted, ethanol-precipitated, and analyzed on 8% acrylamide, 6 M urea sequencing gels.
Electrophoretic Mobility Shift Assays
Binding reactions and electrophoresis were run as described in
Ref. 18. Unless mentioned otherwise, ~30,000 cpm of naked DNA or
reconstituted nucleosomes (about 10 fmol) were combined in a 20-µl
reaction containing 20 mM HEPES, pH 7.4, 80 mM
NaCl, 1 mM EDTA, 3% glycerol, 0.5 µg of salmon sperm
DNA, and 1 to 5 pmol of purified His6-hRAR and/or
F-hRXR
. Complexes were resolved on a 4% native polyacrylamide gel
in 0.5 × TBE at 4 °C at 20 volts/cm (8-10 mA), or when
mentioned on 1% agarose gels in 0.5 × TAE buffer (10 V/cm, 35 mA
for 3 h at 4 °C). 1 × TAE is 90 mM Tris
acetate, pH 7.4, 2 mM EDTA. Antibodies used in supershift
experiments were those used for Western blot analysis of receptor
preparations. Gels were dried and autoradiographed at
80 °C.
Other Techniques
Western Blotting Procedure--
Proteins were resolved on a 10%
SDS-PAGE and transferred onto a nitrocellulose membrane.
Immunodetection of His6-hRAR and F-hRXR
was performed
as described previously using the IBI BioMax system (19).
Protein Assay-- The protein content of receptor preparations was assayed by the Bradford assay (34) using bovine serum albumin as a standard.
Sequencing Reactions--
Sequencing reactions were run using
32P-labeled primers and dideoxynucleotides mixes according
to the manufacturer's instructions (Amersham/U. S. Biochemical
Corp.). Sequencing reactions were run using the native pPro-RARE
plasmid as a template, yielding DNA ladders extending beyond the 5' and
3' ends of the DNA fragment used in reconstitution experiments.
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RESULTS |
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Nucleosome Assembly on DNA Fragments from the Promoter of the Human
RAR Gene--
In this study, we used purified core histones from
HeLa cells (Fig. 1, A and
B) to reconstitute nucleosome core particles following
dilution from high salt. As shown by SDS-PAGE fractionation of core
histones, histone H1 concentration was less than 3% of total histones
(Fig. 1A). Histone oligomers were further characterized by
cross-linking to characterize the octameric structure of purified histones. The main cross-linked band appeared to be an octamer, indicating that our starting material for reconstitution experiments is
indeed a stable core nucleosome. DNA fragments used in nucleosome reconstitution experiments contain a RARE organized as a direct repeat
of two hexanucleotides separated by 5 bp (DR5). An imperfect DR5 RARE
(DR5) is also found 14 bp upstream of this RARE, whereas the TATA box
is located 6 bp downstream of the DR5 sequence. Additional cis-acting
elements are also present in this sequence (Fig. 4). Labeled DNA
fragments of varying length (326, 240, and 182 bp, Fig. 1C)
containing these functional cis-acting elements were used to
investigate whether they can assemble into a nucleosome core in the
presence of histone octamers. Similar amounts of each DNA fragment were
thus used as a template for nucleosome assembly with increasing amount
of histones. As expected, DNA fragments above 200 bp generated
complexes displaying discrete electrophoretic mobilities, indicative of
the association of several histone octamers on these DNAs, or of
distinct octamer positioning (Fig. 1C). More interestingly,
the 182-bp fragment showed a strong propensity to form a unique, low
mobility complex in similar conditions.
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Translational and Rotational Phasing of the Reconstituted
Mononucleosome--
To examine whether this product has features of
native nucleosomes, we first optimized reconstitution parameters to
obtain >95% of the probe reconstituted in nucleosome cores by
carefully adjusting the histone:DNA mass ratio, which was found to be
optimal in the 0.8 to 3.0 range in our conditions (Fig.
2A). Boundaries of the core
particle were determined by the exonuclease III protection assay (Fig.
2B). This enzyme digests from the 3' end to the 5' end of
DNA and its progression along the sequence is strongly, but not
totally, inhibited by proteins bound to DNA. Thus translational positioning of the reconstituted nucleosome can be analyzed by the
ExoIII assay (Fig. 2B). The protection pattern of the
labeled top strand indicated a major histone-induced ExoIII stop at
position +32 (see Fig. 4 for sequence numbering), with weaker
protections being observed at positions +42, +52, + 62. On the
contrary, no stop to ExoIII progression could be detected for the
5'-labeled lower strand, suggesting that histone octamers position
preferentially at the 5' end of this particular DNA fragment. Thus
these data define a ~150-bp segment on which nucleosome cores adopt a
preferential, but not unique, positioning. The nucleosomal structure of
this complex was further characterized by DNase I experiments (Fig. 3). DNase I cleavage sites (which
correspond to a maximal accessibility of the DNA minor groove) on
reconstitutes were clearly different from these of naked DNA. A
periodic pattern extended from 101 to +2 on the upper strand, but the
61 to
20 segment was consistently found to be less accessible to
DNase I cleavage for reasons that are not clear to us. Thus DNase
I-generated fragments were amplified by a linear polymerase chain
reaction (Fig. 3B) to fully characterize the rotational
positioning of the double helix over the DR5 RARE and TATA box
sequences. Again, a highly 10-11 bp periodic cleavage pattern was
observed, typical for a DNA fragment organized around the surface of a
histone octamer. This repetitive pattern was also clearly and
reproducibly detected on the bottom strand in reconstituted DNA
fragments on a stretch extending from
87 to +24 (Fig. 3C).
Thus the DNase I pattern, together with 5' and 3' boundaries defined by
the ExoIII protection assay, strongly suggest that a histone octamer is
positioned from
112 to +32, with the DR5 RARE lying across the dyad
axis of the nucleosome and demonstrate that this precise DNA segment
has an intrinsic structure directing a precise translational and
rotational positioning of the histone octamer. These features are
summarized in Fig. 4.
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Importance of Histones NH2 Termini in RAR/RXR
Heterodimer Binding to Nucleosome Core Particles--
To test this
hypothesis, full-length, E. coli-expressed hRXR and
hRAR
were purified to homogeneity using a
Ni2+-nitrilotriacetic acid affinity matrix (Fig.
5A) and their ability to bind
to a 20-mer oligonucleotidic probe containing the DR5 RARE was assessed
by the electrophoretic mobility shift assay (Fig. 5B).
Cooperative binding of purified RXR/RAR heterodimers to this response
element was consistently observed in these conditions, indicating that
a large fraction of the purified polypeptides is functional and binding
to the core RARE with an affinity in the nanomolar range. Binding of
purified heterodimers to the DR5 RARE present in the 182-bp DNA
fragment used in mononucleosome reconstitution experiments occurred
with a similar efficiency (Fig. 6,
lanes 1-7). The nature of each complex was characterized by
supershift experiments using monoclonal antibodies directed against the
NH2-terminal tag of each receptor. The electrophoretic mobility of DNA·RAR/RXR complexes was clearly decreased in the presence of each immunoglobulin (lanes 8 and 9),
demonstrating that RAR/RXR heterodimers are indeed formed in these
conditions. This DNA template was therefore assembled into a nucleosome
core and tested similarly for its ability to bind RAR/RXR heterodimers. As predicted from the rotational orientation of both half-sites, this
template did not accommodate RXR/RAR dimers, even in conditions where
more than 90% of the naked probe was bound (compare lane 5 to lane 14). 10-Fold higher receptor concentrations were
also used and failed to evidence an interaction of these nuclear
receptors with nucleosomal templates (data not shown). We conclude from these experiments that the organization of retinoic response elements around an histone octamer prevents, in our system, the binding of
hRXR
/hRAR
heterodimers to a prototypic response element.
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DISCUSSION |
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The design of an in vitro system using purified
components with precisely characterized features enabled us to study
whether the association of RAREs with histones is of major importance in regulating the access of retinoid receptors to their cognate DNA-binding sites. We established first that histone octamers are
positioned at a preferential site on the RAR-2 promoter DNA. Boundaries determined by exonuclease III protection experiments located
the nucleosome between
112 and +34, showing that this DNA fragment
has structural properties facilitating DNA bending and therefore
curving around the histone octamer. DNase I protection experiments
carried out on the wild type DNA reconstituted around native core
histones evidenced a typical cleavage pattern alternating every 10 ± 2 bases pairs, and allowed the determination of the rotational
setting of the wild type DNA. Minor grooves of both half-sites were
found to be oriented facing away from the octamer, forming a closed
recognition interface for RXR/RAR heterodimers. Indeed, Rastinejad and
colleagues (35) reported that RXR/thyroid hormone receptor (T3R) DBDs
dimers, and by extension RXR/RAR dimers, engage the major grooves of
the successive half-sites. In contrast, the minor groove of the TATA
box was found to be sensitive to DNase I digestion, evidencing a proper
exposure of the TATA-binding protein-binding site (40). However, this
translational and rotational setting of the TATA box has proven to
provide a poor substrate for TATA-binding protein binding within the
Xenopus borealis 5 S rRNA gene (41) and to the adenovirus
major late promoter TATA box (8). In keeping with these observations,
we found that the nucleosome-assembled DR5 response element did not
allow cooperative binding of RXR/RAR dimers, in opposition to naked
DNA. Glucocorticoid receptor is known to bind to its cognate response
elements as a homodimer structurally close to RXR/T3R and RXR/RAR DBDs
dimers. Wrange and colleagues (25) reported that a glucocorticoid
response element having a analogous rotational setting (with minor
grooves pointing away from the histone octamer) is unable to bind
glucocorticoid receptor dimers. By analogy, we would predict that
modifying the rotational setting of the DR5 RARE would render the
nucleosome permissive to RXR/RAR binding. RXR/T3R dimer binding to the
DR4 thyroid response element of the T3R(
)A promoter forming a
nucleosomal template has been reported to occur both in vivo
and in vitro (42). RXR/T3R binding was, however, in this
system, thyroid-hormone independent; ligand binding led to an
alteration of the chromatin structure and coincidental transactivation
of the promoter. Alteration of the rotational setting of the wild type
T3RE was found be detrimental for the binding of RXR/T3R heterodimers
to nucleosomal DNA (43). Ozato and colleagues (1, 44) reported a
stringent ligand-dependent occupancy of the RARE of the
RAR-
2 promoter in vivo, as well as other cis-acting
elements of the promoter. This occupancy occurred coincidentally to
promoter activation, without inducing major remodeling of the
nucleosomal structure of this promoter. This observation is in striking
contrast with in vitro assays showing that RXR/RAR dimers
bind constitutively to RAREs (i.e. in a ligand-independent manner) (for a review, see Ref. 21, and references therein). We
postulate that, based on our observations, these discrepancies could be
explained by the specific architecture of the RAR-
2 promoter leading
to a closed conformation of the DR5 element in a nucleosomal context,
although its in vivo organization remains to be precisely
characterized.
If RXR/RAR heterodimers are excluded from nucleosomal DNAs in our system, then the nucleoprotein structure of this locus has to be altered in some way to allow for RXR/RAR recruitment. Histone hyperacetylation is the most common post-translational modification targeted to the NH2 termini of these proteins. The reversible charge neutralization of highly conserved lysine residues by acetyl groups reduces the capacity of histones tails to stabilize the path of DNA along the octamer core. Consequently, allosteric changes are thought to occur in the nucleoprotein complex and renders the nucleosomal DNA more accessible to transcription factors (39), although a role in the disruption of nucleosome-nucleosome interactions can be predicted from recent crystallographic data (45). Histone tails clipping in vitro by limited trypsin treatment mimicks the effects of hyperacetylation and facilitate GAL4, TATA-binding protein, and TFIIIA binding to "chromatinized" DNA (39, 41, 46). To ascertain whether this post-translational modification could influence RXR/RAR heterodimers binding to nucleosomal DNA, we used both trypsinized histones and histones extracted from sodium butyrate-treated cells in reconstitution experiments (Fig. 6). Our results demonstrate that both sources of core histones yielded nucleosome core particles able to bind receptor heterodimers, identifying histone hyperacetylation as a major control event in the regulation of retinoid receptors access to DNA. We note that the yeast coactivator Gcn5p has been identified as the catalytic subunit of a histone acetyltransferase (HAT) (47). More relevant to the studied phenomenon is the characterization of a mammalian Gcn5 homologue, p/CAF (48). This protein has been shown to interact with CBP/p300, a co-integrator interacting with T3R, RAR, and RXR (49) and to stimulate the transcriptional activity of progesterone and estrogen receptors (50). CBP/p300 is also a coactivator for a number of transcription factors such as cAMP response element-binding protein, AP-1, MyoD, and c-myb (51-53). Very importantly, CBP/p300 has been shown to possess intrinsic HAT activity (54, 55), suggesting that RXR/RAR binding to RAREs could direct HAT(s) to retinoic acid-regulated promoters. Although transcriptional synergy with CBP/p300 is generally characterized using transiently transfected templates for which the importance of the chromatin structure is questionable (56), this is not exclusive of its involvement in the coordinate recruitment of transcriptional regulators altering chromatin structure. Two other nuclear receptor coactivators, SRC-1 and ACTR, have also been shown to possess histone acetylase activity (57, 58). Thus several HAT activities may be tethered to hormonally-regulated promoters by liganded receptors and act synergistically. A possible cooperativity between two distinct chromatin-modifying complexes is underlined by the physical interaction between the ADA5·Gcn5 complex and the SWI·SNF complex (36), which potentiates glucocorticoid receptor-mediated transcription in yeast (59) and for which mammalian counterparts have been identified (60). Consistent with these observations, the histone deacetylase HDAC1 was found to be associated with SMRT, a nuclear receptor corepressor binding to unliganded RARs (61), and inhibition of cellular deacetylase activities by trichostatin A led to potentiation of retinoic acid-induced transcription and cellular differentiation (62, 63). All the studies above therefore establish a direct link between nuclear receptor-mediated transcriptional activation and histone acetylation.
Several predictions can be made from these and our data, assuming that
retinoid-controlled promoters are organized in nucleosomal arrays: (i)
histone hyperacetylation will be of more or less importance to retinoic
acid-induced transcription activation, depending on the rotational and
translation positioning of the response element. As a consequence of
the observed in vitro positioning of the RAR-2 promoter,
we would predict a strong dependence of its transcriptional activity on
HAT activities. (ii) CBP and/or associated HAT (p/CAF, Gcn5p
homologues) and other factors yet to be identified are likely candidates for chromatin-dependent RXR/RAR coactivators.
Transcription factors like E1A (59), or agents (anti-AP1 retinoids
(64)) known to modulate CBP-retinoid receptors interaction may have a
direct influence on HAT activities targeted to retinoid-controlled promoters, and thus on RA-mediated transcriptional activity. Further experiments in progress in our laboratory comparing the in
vitro and in vivo organization of RA-controlled
promoters will provide new insights into the importance of chromatin
remodeling in retinoid receptors-mediated transcriptional activation,
in conjunction with the use of powerful molecular tools such as
specific ligands and mutated receptors.
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FOOTNOTES |
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* This work was supported in part by grants from the Institut National de la Santé et de la Recherche Médicale, Association de la Recherche sur le Cancer, Fédération Nationale des Centers de Lutte contre le Cancer, and the Université de Lille II. INSERM U459 is part of INSERM IFR22.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Laboratory of Molecular Growth Regulation,
National Insitutes of Child Health and Human Development, NIH, Bldg. 6, Rm. 2A11, Bethesda, MD 20892-2753.
§ Supported by the Ligue Nationale contre le Cancer.
¶ Supported by a fellowship from Association pour la Recherche sur le Cancer.
1 The abbreviations used are: bp, base pair(s); RARE, retinoic acid response element; RAR, retinoic acid receptor; RXR, 9-cis-retinoic acid receptor; DR5, direct repeat with a 5-bp spacer; T3, thyroid hormone; T3R, thyroid hormone response element; CBP, cAMP response element-binding protein; HAT, histone acetyltransferase; PAGE, polyacrylamide gel electrophoresis; ExoIII, exonuclease III.
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