Retinoid Receptor-Induced Alteration of the Chromatin Assembled on a Ligand-Responsive Promoter in Xenopus Oocytes
Saverio Minucci1,
Jiemin Wong,
Jorge C. G. Blanco,
Yun-Bo Shi,
Alan P. Wolffe and
Keiko Ozato
Laboratory of Molecular Growth Regulation (S.M., J.C.G.B., K.O)
Laboratory of Molecular Embryology (J.W, Y.-B. S, A.P.W) National
Institutes of Child Health and Human Development Bethesda, Maryland
20892
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ABSTRACT
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Retinoic acid (RA) stimulates transcription from
the retinoic acid receptor ß2 (RARß2) promoter in mammalian
embryonal cells. Evidence by in vivo deoxyribonuclease I
(DNase I) hypersensitivity assay indicates that RA treatment of these
cells results in an alteration of chromatin structure in and near the
promoter. To study the role of chromatin in RA-activated transcription,
we assembled the RARß2 promoter into chromatin in Xenopus
oocytes. Ectopic expression of RAR and retinoid X receptor (RXR)
enhanced transcription without ligand, irrespective of whether
chromatin was assembled in a replication-dependent or -independent
manner, although ligand addition led to a further, marked increase in
transcription. Moreover, expression of RAR and RXR, without ligand
addition, induced DNase I-hypersensitive sites in the
chromatin-assembled promoter. Futhermore, expression of RAR and RXR in
oocytes led to local disruption of chromatin assembled over the
promoter without ligand. Similar ligand-independent, but
RXR/RAR-dependent nucleosomal disruption was observed in an in
vitro chromatin reconstitution system using
Drosophila embryonic extracts. Thus, unliganded receptors
expressed in oocytes are capable of accessing to the
chromatin-assembled promoter and activating transcription without
ligand, indicating that chromatin assembly per se is not
sufficient to reproduce ligand-dependent chromatin changes and promoter
activation seen in mammalian cells. The oocyte system may serve as a
model to study mechanisms of RA-dependent alterations of chromatin
structure.
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INTRODUCTION
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Retinoic acid receptor ß2 (RARß2) is one of the immediate
early genes induced by retinoic acid (RA) in mouse and human embryonal
carcinoma cells (1, 2, 3, 4, 5) and is thought to play a role in subsequent
elicitation of biological effects (6, 7). The promoter is active in
early mammalian embryos (8) and myeloid cells (9). Analyses of cells
with disrupted RAR genes indicate that transcription of the RARß2
promoter is mediated by multiple RARs (10). Both the human and mouse
RARß2 promoters contain, within a 150-bp upstream region, a canonical
RA-responsive element of the DR-5 type, to which the retinoid X
receptor (RXR)/RAR heterodimer binds (1, 2, 4, 5). Additional elements
present within this region are a cAMP-responsive element and an
auxiliary retinoic acid response element (RARE), both of which affect
RA-responsive promoter activity (3, 4, 11). We have recently studied
RA-induced chromatin structure alterations in and near the RARß2
promoter in P19 embryonal carcinoma cells and found that this region of
chromatin undergoes a rapid change after RA addition, as evidenced by
induction of restriction site accessibility and deoxyribonuclease I
(DNase I) hypersensitivity, which coincides with transcriptional
activation of the promoter (12). These changes also correlated with
ligand-induced in vivo footprinting of this promoter (4, 5, 9). These findings suggested that the chromatin structure formed in and
around the RARß contributes to transcriptional activity. Of further
interest, RA-induced restriction site accessibility and DNase I
hypersensitivity seen in this promoter are remarkably similar to
glucocorticoid- and progesterone-induced chromatin changes in the mouse
mammary tumor virus (MMTV) promoter, which have been extensively
studied in various cells both with integrated and episomal promoters
(13, 14, 15, 16). The similarity is extended to ligand-dependent appearance of
footprints in vivo (17). Although transcriptional activation
coincides with extensive nucleosomal disruption in some genes (18, 19, 20),
ligand-dependent changes in the MMTV and RARß2 promoters do not seem
to accompany gross reorganization or disruption of nucleosomes over the
promoter (12, 16, 21).
The Xenopus oocyte chromatin assembly system (22, 23, 24, 25) offers
a versatile model to study how chromatin affects transcription. A
significant advantage of this system over in vivo cell
systems is that the chromatin assembly and transcription can be studied
with exogenously injected templates and transcription factors expressed
from injected RNAs. Additionally, with this system, one can study the
activity of chromatin that had been assembled during or after template
replication (22). The oocyte system has been used for analysis of
chromatin assembly and transcription of the Xenopus TRßA
gene (23, 24). These studies demonstrated that in the absence of
thyroid hormone (T3), RXR/(thyroid hormone receptor (TR)
heterodimers efficiently bind to the promoter assembled in nucleosomes
and repress transcription. T3 addition results in
transcriptional activation and extensive disruption of nucleosomes,
although chromatin disruption by itself is not sufficient for
transcriptional activation (24). These findings provided a valuable
reference to the present study, which addressed RA-responsive
transcription by RXR/RAR in the oocyte injection system. In this study
we asked 1) whether ectopically expressed RXR/RAR heterodimers can
access to a chromatin-assembled RARß2 promoter and induce changes in
its structure, and 2) whether ectopically expressed RXR/RAR
heterodimers repress and enhance transcription depending on ligand
addition, as observed with the TRßA promoter (23, 24).
Our results show that unliganded RXR/RAR heterodimers are capable of
accessing to the chromatin-assembled promoter and inducing DNase I
hypersensitivity in a position comparable to that induced in P19 cells
after ligand addition. Furthermore, expression of unliganded receptors
led to disruption of nucleosomes as detected by altered sensitivity to
micrococcal nuclease (MNase) digestion. In accordance, unliganded
heterodimers stimulated transcription from the RARß2 promoter without
requiring ligand, although ligand addition markedly increased levels of
transcription. No evidence for transcriptional repression was observed
in the RA-responsive promoter. Thus, the RARß2 promoter can be
efficiently assembled into chromatin to direct transcription by the
unliganded heterodimer, suggesting that ligand requirement observed in
the natural environment is ascribed to another level of regulation.
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RESULTS
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Expression and Function of Retinoid Receptors in
Xenopus Oocytes
As a first step in the analysis of the function of retinoid
receptors in the chromatin assembly system, oocytes injected with
capped mRNAs encoding the Xenopus RXR
and RAR
1 (27)
were tested for RARE-binding activity by electrophoretic mobility shift
assays (EMSA). As shown in Fig. 1
, A and
B, extracts from uninjected oocytes showed no detectable binding
activity for the RARE derived from the RARß2 gene (1, 2, 4, 5).
Injection of increasing amounts of mRNAs for RAR and RXR led to a
dose-dependent increase in the formation of a DNA-protein complex (Fig. 1A
, lanes 24), which was competed by excess unlabeled RARE (Fig. 1A
, lane 5). The complex was composed of RXR/RAR heterodimers, since it was
detected after injection of mRNAs for both RAR and RXR, but not of a
single mRNA for either receptor alone (Fig. 1B
, compare lanes 2 and 3
with lanes 4 and 5). The heterodimer-RARE complex was observed even
when oocyte extracts injected with single mRNA were later admixed (Fig. 1B
, lane 5). Thus, injection of the two mRNAs into Xenopus
oocytes leads to the formation of heterodimers that bind to the
RARE.

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Figure 1. Expression of RXR and RAR in Xenopus
Oocytes after mRNA Injection
A and B, EMSA analysis: in panel A, oocyte extracts from uninjected
oocytes (lane 1) or oocytes injected with 0.02, 0.2, and 2 ng of mRNAs
for RXR and RAR (lanes 24) were subjected to EMSA using the 188-bp
RARß2 promoter fragment as a probe, in the absence (lanes 14) or in
the presence (lane 5) of 50-fold excess unlabeled RARE oligomers. In
panel B, extracts from uninjected oocytes (lane 1) or oocytes injected
with 2 ng of single mRNA for RXR (lane 2) or RAR (lane 3) or 1 ng each
of both mRNAs (lane 4) were tested. In lane 5, extracts from oocytes
injected with a single mRNA for RAR or RXR were mixed and tested as
above. The arrow indicates the position of heterodimers.
The identity of the faster migrating band of weak intensity has
not been determined. C and D, Transcription analysis: oocytes nuclei
were injected with 0.2 or 2 ng of the ds-RARß2/GL3 plasmid harboring
the wild-type RARß2 promoter (nt -65 to nt +14, containing one
RARE), or xRARE/GL3 (the promoter with a point mutation in the RARE
that abolishes receptor binding (C) and G6PD-luc (harboring the murine
G6PD promoter) (D), followed by intracytoplasmic injection of mRNAs for
RXR and RAR (0.2 ng RNA for lanes 1 and 4 in panel C, 0.02 ng RNA for
lane 2 in panel C, and 2 ng RNA for lanes 3 and 4 in panel D). The
oocytes were incubated with all-trans-RA at 10
µM for 1215 h, and luciferase transcripts were detected
by primer extension.
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To determine whether exogenously expressed receptors stimulate
transcription, oocytes were injected with the receptor mRNAs and the
double-stranded (ds) plasmid DNA harboring the RARß2 promoter
connected to the luciferase gene (4, 5). Oocytes were then treated with
10 µM all-trans-RA for 12 h, and the
levels of luciferase RNA were measured by primer extension (Fig. 1C
).
In the mock injection, only very low levels of luciferase RNA were
detected (Fig. 1C
, lane 3). However, injection of receptor RNAs led to
a dose-dependent, 20- to 30-fold increase in reporter expression (Fig. 1C
, lanes 1 and 2). In contrast, the mutated RARß2 promoter
containing a point mutation in the RARE that abolished binding of
RXR/RAR heterodimer (5, 28) failed to increase reporter expression
(Fig. 1C
, lanes 4 and 5). Dot-blot analysis of the DNA extracted from
the oocytes shows that similar amounts of reporter DNA were injected,
confirming the validity of transcriptional activation after receptor
mRNA injection (Fig. 1C
). To establish the specificity of
transcriptional stimulation, an RA-nonresponsive glucose-6-phosphate
dehydrogenase (G6PD) promoter (29) connected to the luciferase gene was
also tested. As shown in Fig. 1D
, levels of luciferase RNA were
unaffected by injection of the mRNAs, either in the presence or absence
of RA. These results show that the RXR/RAR heterodimer expressed in
Xenopus oocytes binds to the RARE and enhances transcription
from an RA-responsive promoter.
RXR/RAR Heterodimers Stimulate Transcription from Both the
Single-Stranded (ss) and ds RARß2 Templates: The Absence of
Repression by Unliganded Heterodimer
One of the advantages of the oocyte system is that ss-DNA
templates undergoing replication can be assembled into nucleosomes
(22). Chromatin assembly on a ss-template follows kinetics faster than
those on a ds-template, and the resultant template represses basal
transcription more efficiently than a ds-DNA template. Previous work
(23, 24) showed that transcription from the TRßA promoter derived
from the ss-DNA template is strongly repressed by unliganded TR and
RXR, and the repression is relieved by addition of T3.
Thus, it was of interest to test whether RAR and RXR expressed in
oocytes act in a comparable manner on the RARß2 promoter. In Fig. 2A
, transcription from the RARß
promoter was examined on the ss-template. To test the effect of the
timing of receptor expression, the order of injection was varied with
respect to the template DNA and receptor mRNAs; in protocol a, oocytes
were first injected with the ss-template, incubated for 6 h
(allowing replication-coupled chromatin assembly to complete) (22), and
then they were injected with the receptor mRNAs. In protocol b, oocytes
were first injected with the receptor RNAs, incubated for 6 h
(allowing sufficient levels of RXR and RAR expression), and then
injected with the DNA template. In experiment a, newly synthesized
receptors would have to access to the RARE on a fully assembled
template to exert their activity, whereas in experiment b,
presynthesized receptors might have access to the RARE during chromatin
assembly. As shown in Fig. 2A
(lanes 1 and 2), without injected
receptor mRNAs, ss-DNA template produced very low levels of
transcripts. Addition of RA did not significantly increase
transcription; depending on the batches of oocytes, a low level of RA
inducibility was occasionally found, presumably due to the low level
expression of endogenous receptors (data not shown). However, injection
of receptor mRNAs significantly increased transcription even in the
absence of ligand (lanes 3 and 5), which was observed regardless of
whether the template was injected before or after mRNA injection.
Transcriptional repression was not observed by the unliganded receptors
under these conditions. Furthermore, addition of RA caused a marked
increase in transcription (Fig. 2A
, lanes 4 and 6), again regardless of
the order of injection (compare lanes 3 and 4 with lanes 5 and 6).
These results show that unliganded RAR and RXR stimulate RARß2
transcription from the template that was assembled into chromatin
during replication, and addition of ligand only increases levels of
transcription. Transcription of the G6PDH gene (23), measured as a
control, was comparable in these oocyte samples (Fig. 2A
). The effect
of ligand was also tested on transcription from a ds-RARß2 template.
As shown in Fig. 2B
, without receptor mRNA injection, transcription was
minimal in the absence of ligand, but addition of RA led to some
transcription in these samples, which could be attributed to the low
level expression of endogenous receptors in oocytes (lanes 1 and 2).
Similar to the results with the ss-DNA template, injection of receptor
mRNAs stimulated transcription from the ds-template in the absence
of ligand, and addition of RA further increased transcription (Fig. 2B
, lanes 36). Again, no transcriptional repression was seen by
unliganded receptors. The outcome was the same, irrespective of whether
receptor mRNAs were injected before or after the injection of the
template. The transcript levels of the endogenous histone (H4) gene
(23), tested as a control, were comparable in the these oocyte samples
(Fig. 2B
).

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Figure 2. Functional Analysis of RXR/RAR Heterodimers in
Xenopus Oocytes
A, Oocytes were injected with the ss-plasmid RARß2/GL2 containing the
wild-type RARß2 promoter (nt -104 to nt +14) and the ds-G6PD-luc
plasmid. In lanes 34, oocytes were injected with 0.2 ng of receptor
mRNAs 6 h before injection of plasmid DNA. In lanes 56, oocytes
were injected with DNA 4 h before injection of the mRNAs. Oocytes
were incubated with (+) or without (-) 10 µM
all-trans-RA for overnight and luciferase RNA was
detected by primer extension. B, Oocytes were injected with the
ds-plasmid RARß2/GL2. In lanes 34, oocytes were injected with 0.2
ng of receptor mRNAs 6 h before injection of plasmid DNA. In lanes
56, receptor mRNAs were injected 6 h after plasmid DNA
injection. Luciferase transcripts were detected as above. As a control,
transcripts for endogenous H4 were detected by primer extension. C,
Oocytes were injected with the ds-plasmid RARE2tk-luc (containing two
copies of the RARE in front of the tk promoter fused to the luciferase
gene) 68 h before injection of 0.2 ng of receptor mRNAs.
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To examine whether transcription mediated by RXR and RAR is influenced
by the context of the basal promoter, we tested another ds-template in
which two copies of the RARE (DR-5) were fused to the herpes simplex
virus thymidine kinase (TK) promoter (5, 28). As shown in Fig. 2C
, in
the absence of receptor mRNA injection, transcription was low but was
increased after addition of RA, analogous to the RARß2 promoter
above. Similarly, injection of receptor RNAs greatly increased
transcription even in the absence of RA, although RA gave an additional
increase in transcription. As in Fig. 2B
, transcript levels of control
histone H4 were comparable in these samples. These results indicate
that both unliganded and ligand-bound RXR/RAR heterodimers stimulate
transcription from RA-responsive promoters, regardless of whether the
chromatin was assembled during or after DNA replication. As detailed in
Discussion, these results are similar to the observations
made in an in vitro transcription system (11).
Binding of RXR/RAR Heterodimers to the Nucleosome-Associated
RARß2 Promoter in Vitro
The above experiments suggest that RXR/RAR heterodimers are
capable of accessing to the retinoid-responsive promoters assembled in
oocyte chromatin. To ascertain the accessibility of the heterodimers to
the chromatin-associated template in vitro, EMSAs were
performed with a 188-bp RARß2 promoter fragment complexed with
monosomes and recombinant RXR/RAR heterodimers. The fragment prepared
by PCR encompassed from nucleotide (nt) -128 to nt +60 of the RARß2
promoter, which includes the main RARE (-51 to -33) and the auxiliary
RARE (-79 to -61) (11). This fragment was assembled into nucleosomes
using histone octamers prepared from chicken erythrocytes (30). As
shown in Fig. 3A
(lanes 14), incubation
of free DNA fragment with increasing concentrations of heterodimers led
to the formation of multiple complexes representing binding to the two
RAREs. Two bands seen in lane 3 likely represent heterodimer binding to
one or two RAREs. As shown in Fig. 3A
(lanes 59), the
monosome-associated DNA fragment without receptors migrated to position
b (compare free DNA that migrated to position a). Addition of
increasing amounts of RXR/RAR heterodimer led to the formation of more
slowly migrating bands (shown as df). The formation of receptor-DNA
complexes in monosome-assembled promoter was observed with
approximately 10-fold higher amounts of heterodimers than in free DNA.
Bands df probably represent heterodimers complexed with the
monosome-assembled promoter, whereas band c may represent heterodimers
bound to a small amount of residual free DNA. The heterodimer-monosome
DNA complexes (d and e) were eliminated by excess RARE oligomers of the
DR-5 type, but not control oligomer (Fig. 3B
). As expected from EMSA
data with an RARE probe (Fig. 1
), addition of RA did not affect the
complex formation (data not shown). These results show that the
monosome-bound RARß2 promoter permits binding of the RXR/RAR
heterodimer through the RAREs in the absence of ligand.

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Figure 3. EMSA Analysis with a Monosome-Bound RARß2
Promoter in Vitro
An end-labeled 188-bp RARß2 promoter fragment (-128/+60) generated
by PCR was assembled into nucleosomes in vitro. A,
Increasing amounts of purified recombinant RXR and RAR were incubated
with a free RARß2 fragment (lanes 14) or with a fragment
reconstituted into a monosome (lanes 59) and analyzed on a 4% native
polyacrylamide gel. The free fragment is marked by a; monosome-bound
DNA fragment without receptors is marked by b. Heterodimer/DNA
complexes are shown as cf. B, Competition analysis. A free (lane 1)
or monosome-bound RARß2 fragment was incubated with RXR and RAR in
the absence (lane 2) or presence of 34-bp RARE oligomers (lane 3) or
control oligomers of the YY1-binding site (lane 4). b Represents
monosome-bound DNA. c Is likely heterodimers bound to free DNA. df
Represent heterodimer-monosome-DNA complexes.
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Heterodimer-Induced Chromatin Alterations in Xenopus
Oocytes
Oocyte injection data in Figs. 1
and 2
and binding data in Fig. 3
indicate that the chromatinized template is accessible to receptors
both in the absence and in the presence of the ligand. Previously,
however, an alteration of chromatin structure was observed in and
around the RARß2 promoter in a RA-dependent manner in mouse embryonal
cells (12). A similar hormone-dependent chromatin structure transition
has been noted for the MMTV promoter in various cells (13, 14, 16, 17).
Thus, it was important to assess whether the chromatin assembled in
Xenopus oocytes undergoes a structural alteration after
heterodimer expression and whether such changes, if observed, are
ligand dependent. Oocytes were injected with the ss-RARß2 template
before or after injection of receptor mRNAs. Oocytes were then
homogenized and digested with increasing concentrations of DNase I. DNA
was purified, cut with EcoRI in vitro, and
subjected to Southern blot hybridization with a
32P-labeled, 600-bp HindIII-EcoRI
fragment as a probe. As shown in Fig. 4
(lanes 18), injection of the template alone generated several diffuse
bands of low intensity, and the addition of RA did not affect the
digestion pattern. However, injection of receptor mRNAs led to the
appearance of two hypersensitive bands, which mapped within the RARß2
promoter (arrows in Fig. 4
). The appearance of the
hypersensitive bands was observed both when template was injected
before and after mRNA injection and was not affected by addition of RA.
Digestion of purified plasmid DNA presented in lanes 25 and 26 shows a
uniform distribution, indicating that the patterns observed with the
oocyte samples are not due to intrinsic sensitivity of the promoter to
DNase I. Thus, heterodimer expression leads to the generation of DNase
I-hypersensitive sites in the RARß2 promoter, indicating that the
chromatin structure undergoes a marked change upon binding of receptors
to the template. Similar sites in the RARß2 promoter were found to be
sensitive to DNase I in P19 cells, but were detected in a RA-dependent
manner (12)

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Figure 4. Induction of DNase I Hypersensitivity by RXR/RAR
Heterodimers
Oocytes were injected with a ss-RARß2/Gl2 plasmid. In lanes 916,
they were injected with 0.2 ng of receptor mRNAs before injection of
plasmid DNA. In lanes 1724, oocytes were injected with plasmid DNA
before injection of receptor mRNAs. After overnight incubation with 10
µM all-trans-RA, oocyte homogenates were
digested with DNAseI (0.2 to 2 U). Purified DNAs were digested with
EcoRI in vitro, resolved on an agarose
gel, blotted, and hybridized with an end-labeled
EcoRI/HindIII probe (shown in the
bottom). Plasmid DNA (lanes 25 and 26) was digested with
DNaseI and EcoRI and run as a control. The
arrows indicate the position of the hypersensitive sites
induced after receptor expression.
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Heterodimer-Induced Nucleosomal Disruption in Xenopus
Oocytes
Previous studies with TR/RXR heterodimers showed that nucleosomes
organized over the TRßA promoter are extensively disrupted after
T3 addition even in the absence of transcription (23, 24).
Nucleosomal disruption coincidental with gene activation has been shown
to occur in other promoters as well (18, 19, 20). However, for some
ligand-responsive promoters, nucleosome disruption does not seem to be
readily observed (12, 16, 21). Here we studied whether receptor
expression changes configuration of nucleosomes over the RARß2
promoter assembled in oocytes. Oocytes were injected first with the
ss-RARß2 template, followed by second injection with receptor RNAs
6 h later. Oocyte homogenates were digested with increasing
amounts of MNase, DNA purified, and subjected to Southern blot analysis
with a fragment encompassing the RARß2 promoter (-128/+60). As shown
in Fig. 5A
, oocyte samples injected with
the template alone without receptor generated a MNase digestion pattern
indicative of ordered nucleosomal organization (with the position of
mono-, di-, and trisomes shown by arrowhead). This pattern
was not affected by addition of RA. Oocyte samples injected with
receptor RNAs appeared to be more extensively digested than samples
without receptor injection and produced a distinct band below the size
of a monosome (lanes 46, arrow in Fig. 5A
). Very similar
digestion patterns were observed after treatment with RA (lanes 79).
However, when these samples were probed with a control fragment derived
from the plasmid vector without the RARß2 promoter (Fig. 5B
), the
MNase digestion patterns were essentially the same among uninjected
oocytes and those injected with receptor mRNAs (compare lanes 13 with
lanes 46), and RA treatment did not alter the digestion pattern
(lanes 79). These results suggest that expression of RXR/RAR
heterodimers leads to disruption of local nucleosomes, which is
unaffected by ligand.

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Figure 5. Nucleosomal Disruption Induced by Unliganded
RXR/RAR: Analysis with the Xenopus Oocyte System
Oocytes were injected with the ss-RARß2SK plasmid 3 h before
injection of mRNAs (2 ng) and incubated overnight in the presence or
absence of 10 µM all-trans-RA. Oocyte
homogenates were digested by MNase (2, 6, or 30 U), and purified DNAs
were analyzed by Southern blot hybridization using a probe
corresponding to the RARß2 promoter (nt -128 to nt +60, panel A) or
with the vector plasmid (panel B).
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Heterodimer-Induced Nucleosome Disruption in an in
Vitro Reconstitution System
Nucleosomal disruption by RAR and RXR observed over the RARß2
promoter in oocytes seemed to differ somewhat from that by TR and RXR,
where addition of T3 dramatically increased disruption of
nucleosomes assembled over the TRßA gene (23, 24).
To further study the possible nucleosomal disruption by RXR/RAR
heterodimers, and the effect of ligand, we used an in vitro
chromatin reconstitution system developed by Tsukiyama et
al. (31) that utilizes Drosophila embryo extracts. This
cell-free system has been investigated for the nucleosomal organization
of the hsp70 gene, which led to recent isolation of
nucleosome-remodeling factors (32). In the experiments shown in
Fig. 6
, the RARß2 gene fragment from nt
-630 to nt +570 in the pSK plasmid was reconstituted into chromatin
with the Drosophila extracts to which recombinant RXR and
RAR were added before (lanes 46) or after chromatin reconstitution
(lanes 712). Reaction mixtures were digested with MNase and subjected
to Southern blot analysis with a probe corresponding to the RARß2
promoter (Fig. 6A
) or to a region farther upstream from the promoter,
tested as a control (Fig. 6B
). Similar to the previous observations
with the hsp70 promoter (31, 32), incubation with the
Drosophila embryo extracts, without receptors, led to the
assembly of an ordered nucleosomal array on the RARß2 template (lanes
13 in Fig. 6
, A and B). Addition of RAR and RXR drastically altered
MNase digestion pattern in the RARß promoter region; it generated
extensive subnucleosomal fragments with a concomitant reduction in the
oligosomal and monosomal complexes, which was seen in a MNase
dose-dependent manner (Fig. 6A
, lanes 412). Similar subnucleosomal
fragments were reported to be produced after factor binding to the
hsp70 gene (32). Essentially the same pattern of alterations in the
MNase digestion was observed after RA addition (compare lanes 45 with
lanes 79 in Fig. 6A
), irrespective of whether receptors were added
before or after chromatin assembly. In contrast, the MNase digestion
pattern for the farther upstream region (Fig. 6B
) was not affected by
addition of heterodimers and/or of RA. These results are consistent
with the data in Fig. 5
and show that RXR/RAR heterodimers are capable
of disrupting local nucleosomes assembled over the promoter, which is
not influenced by RA.

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Figure 6. Nucleosomal Disruption Induced by Unliganded
RXR/RAR: Analysis with the in Vitro Chromatin
Reconstitution System
The plasmid RARß2SK containing the RARß2 promoter (from nt -630 to
nt +570) was assembled into chromatin in vitro using
Drosophila embryo extracts (Materials and
Methods). In lanes 46, purified RXR/RAR heterodimers were
added 6 h before assembly, whereas in lanes 712 RXR/RAR was
added 6 h after assembly and incubated in the presence or absence
of 10 µM all-trans- RA for 40 min.
Reactions were digested with 25 U of MNase at room temperature.
Purified DNAs were analyzed by Southern blot hybridization using an
oligonucleotide probe corresponding to the RARE in the RARß2 promoter
(nt -58 to nt -28, panel A) or to the region nt -550 to nt -575
(panel B). The positions of mono-, di-, and trisomes are marked with
open arrowheads.
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DISCUSSION
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The present study found that the RARß2 promoter assembled into
chromatin in Xenopus oocytes drives transcription through
ectopically expressed RXR and RAR. This process coincides with
ligand-independent alteration of local chromatin structure. The
transcription and chromatin changes caused by unliganded RAR/RXR
heterodimers revealed in this system share some features with, but
differ in other aspects from, those observed by TR/RXR heterodimers in
the oocyte model (23, 24) and those in P19 embryonal cells (12).
Transcriptional Activation by Unliganded RXR/RAR Heterodimers: The
Lack of Transcriptional Repression
Expression of unliganded RXR and RAR led to stimulation of
transcription from both the RARß2 and RARE-TK promoters under all
conditions tested, i.e. where the template was preassembled
before receptor expression, or assembled in the presence of receptors,
and where the injected template was a ss- or ds-plasmid (Figs. 1
and 2
). Nevertheless, addition of RA led to a further increase in
transcription in all of these cases. A notable aspect of these findings
is the lack of transcriptional repression by unliganded RXR/RAR, which
might have been anticipated as unliganded TR/RXR repressed
transcription from the TRßA promoter, particularly strongly when the
ss-DNA template was assembled into chromatin, and more weakly with a
ds-template (23, 24). In all cases, repression was found neither with
the RARß2 nor with the RARE-Tk promoter (Figs. 1
and 2
). Repression
by RXR/RAR might also have been expected, based on the previous study
showing that TR and RAR repress transcription from chimeric promoters
transfected into mammalian cells (33, 34).
Although detailed information on retinoid levels in Xenopus
oocytes is not available to us, the lack of repression is not easily
explained by the possible presence of endogenous retinoids, since
addition of RA caused marked transcriptional stimulation, occasionally
even in oocytes that had not been injected with receptor mRNAs (Fig. 2B
), where receptor concentrations were much lower than in the presence
of ectopic receptors. These observations indicate that transcriptional
repression is not an obligatory function of unliganded receptors, but
may depend on specific promoter architecture. Moreover, results of
transcription analysis obtained in the oocyte system bear a remarkable
resemblance to those observed by an in vitro transcription
system described by Valcarcel et al. (11), in which
transcription from the RARß2 promoter was stimulated by the RXR/RAR
even in the absence of ligand, although ligand addition increased
levels of transcription. Thus, the RARß2 promoter assembled in the
oocyte system (and the in vitro transcription system)
appears to obliterate ligand requirement imposed in the native cellular
environment, presumably because the template assembled in this (and the
in vitro transcription) system is readily accessible to the
unliganded RXR/RAR heterodimer, while that in the native environment
requires a ligand-dependent step to bind to heterodimers (4).
Unliganded heterodimers, once gaining access to the template, may
moderately activate transcription perhaps through the AF-1 domain,
and/or ligand independent interaction with basal transcription factors
even in the absence of coactivator recruitment.
Ligand-Independent Alteration of Chromatin: DNase I
Hypersensitivity
In several models, induction of DNase I hypersensitivity is
associated with transcriptional activation (35, 36, 37). DNase I
hypersensitivity is induced in a hormone-dependent manner in the MMTV
promoters and is accompanied by factor occupancy (14, 15, 16). Thus, it is
reasonable to assume that induction of DNase I hypersensitivity by
unliganded RXR/RAR seen in the present work is a consequence of their
binding to the template. Similarly, DNase I-hypersensitive sites are
shown to be induced in the TRßA promoter by unliganded TR/RXR (24);
however, although T3 enhances the levels of
hypersensitivity in the TRßA promoter, RA had no effect on the
RARß2 promoter. In contrast to the apparent lack of ligand effects,
previous analyses of the RARß2-luciferase gene integrated into P19
embryonal cells showed ligand-dependent induction of DNase I
hypersensitivity (12). Interestingly, the hypersensitive sites mapped
in the present study are very close, if not identical, to those mapped
in P19 cells (12). Since the promoter and reporter fragment analyzed
here are the same as that in Ref. 12, and since DNase I
hypersensitivity coincided with ligand-dependent occupancy of the
receptors to the promoter in P19 cells, it is likely that in the oocyte
system unliganded receptors are capable of binding to the template,
bypassing the ligand requirement present in P19 cells. It is possible
that the oocyte system may not contain sufficient levels of a
cofactor(s) that governs ligand-dependent accessibility of receptors.
The absence of ligand dependence may alternatively be attributed to the
properties of histones assembled in this system, such as levels of
acetylation or deacetylation of nucleosomal histones, which are shown
to influence accessibility of transcription factors to the template
(26, 38), or to the availability of linker histones (39, 40). In either
case we feel that chromatin changes found in this system are
unlikely to be attributed to the architecture of the promoter,
i.e. the mini-chromosome rather than an integrated sequence,
since chromatin changes comparable to integrated counterparts have been
reported in the MMTV promoter in the episomal plasmids (14).
Nucleosomal Disruption Induced by Unliganded RXR/RAR
Expression of RXR/RAR heterodimers markedly increased MNase
sensitivity over the RARß2 promoter (Fig. 5
), indicating that binding
of unliganded heterodimers to the promoter leads to nucleosomal
disruption in a locally restricted manner. A comparable local
nucleosomal disruption was observed by unliganded RXR/RAR heterodimers
in an in vitro model of chromatin reconstitution using
Drosophila extracts. This in vitro system has
been extensively analyzed by Tsukiyama and co-workers (31, 32), who
isolated a nucleosome-remodeling factor, NURF, which disrupts
nucleosomes assembled over the hsp70 promoter. It is possible that such
a nucleosome-remodeling factor(s) is recruited by unliganded RXR/RAR in
the oocyte system as well as the in vitro system. Although
RA had no detectable effects on nucleosomal disruption over the RARß2
promoter, T3 dramatically increased chromatin disruption
over the TRßA promoter in the oocyte system (23, 24). At present, the
basis of the different behavior of ligands seen for the RARß and
TRßA promoter is not clear. However, it is interesting to note that
despite conspicuous nucleosomal disruption observed for this and the
TRßA promoter in the oocyte system (23, 24), ligand-dependent
nucleosomal remodeling/disruption has not been readily demonstrated for
the MMTV and RARß2 promoters in mammalian cells in vivo
(12, 16). Considering extensive nucleosomal disruption observed in
other promoters (18, 20), it may be reasonable to assume that
nucleosomal disruption is an integral part of chromatin changes
associated with gene activation, but can be obscured by other causes
for some promoters such as the MMTV and RARß2 in P19 cells: in
vivo nucleosomal disruption may occur only transiently and may not
be easily detected by the standard MNase digestion assay. It is also
possible that other factors mobilized to the promoter may reduce
sensitivity to MNase digestion. In any event, although ligand addition
had no apparent effect on the extent of nucleosome disruption and of
DNase I hypersenstivity, it did enhance transcription from the RARß2
promoter. These results suggest that nucleosomal disruption is not a
consequence of transcription, although it may be a prerequisite for
transcription. Consistent with this notion, nucleosomal disruption
occurred even in the absence of transcription (23). Thus, in the oocyte
system RA might exert its effects on an event that follows receptor-
induced chromatin alterations, but which affects transcription.
In summary, the Xenopus oocyte system employed in this work
shows that unliganded RXR/RAR heterodimers can induce extensive
chromatin alterations over the RARß2 promoter and stimulation of
transcription, bypassing the ligand requirement present in the native
environment. This system might serve as a valuable model to investigate
the molecular mechanisms underlying receptor-induced chromatin
alterations.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Plasmids RARß2SK, RARß2GL2, RARßs/GL3, xRAREs/GL3,
RARE(2)tk-luc, containing the murine RARß2 promoter, and G6PD-luc
have been described (4, 5, 28, 29). The ss-plasmids were prepared from
the phagemids induced with VCS M13 as described (23). mRNAs encoding
Xenopus RXR
and Xenopus RAR
1 mRNAs were
prepared from plasmids kindly provided by Bruce Blumberg (27) using the
Message Machine kit (Ambion, Austin TX) according to the
manufacturers protocol.
Microinjection of Xenopus Oocytes
These procedures were carried out as described (22, 23). The
DNAs (1.1 ng per oocyte for ss-DNA, 2.2 ng per oocyte for ds-DNA) were
injected into the oocyte nuclei, whereas RNAs (amounts indicated in
each assay) were injected into the oocyte cytoplasm. After injection,
the oocytes were incubated at 18 C overnight in buffer supplemented
with antibiotics (22). The amounts of injected DNA were quantified by
dot-blot or Southern blot hybridization of purified DNA (23).
Transcription Analysis
RNAs were prepared from 1520 injected oocytes (15, 16, 17, 18, 19, 20) using
the RNAzol (Tel-test, Inc., Friendswood, TX) as described (23).
Luciferase transcripts were detected by primer extension using the
primer 5'-CCAGGGCGTATCTCTTCATAGCC-3', corresponding to nt position +126
to +149 using the amounts of RNA equivalent to two oocytes. Histone H4
transcripts, measured as an internal control, were detected with the
primer described in Ref. 24.
EMSA Assay with a Nucleosome-Reconstituted RARß2 Probe
A 188-bp fragment from the RARß2 promoter (-128/+60) was
generated by PCR with one of the two primers end-labeled with
32P. The PCR products were purified and reconstituted with
the octamer exchange method as described, using histone octamers
prepared from chicken erythrocytes (23, 30). The monosome fraction was
then purified by sucrose gradient separation as described (30).
Recombinant murine RXRß and human RARß with a histidine tag at the
N-terminal end were produced in a baculovirus vector and purified on
nickel-affinity columns. Their ability to heterodimerize with each
other and to specifically bind to RARE oligomers was verified for each
receptor preparation: the details of vector construction, receptor
purification, and RARE binding activities will be described elsewhere
(J. C. G. Blanco, S. Minucci, X.-J. Yang, K. K. Walker, H. Chen, R. M.
Evans, Y. Nakatani, and K. Ozato, unpublished). EMSA analysis was
performed as described (23).
DNAse I Hypersensitivity Analysis
These analyses were performed essentially as described (24).
Briefly, 20 injected oocytes were homogenized in digestion buffer (10
mM HEPES, pH 8.0, 50 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 0.1% NP40, and 5%
glycerol). Equal aliquots of the extracts (5 oocyte equivalent) were
digested with 2.5, 5, 10, and 20 U of DNAse I (GIBCO-BRL, Bethesda, MD)
for 2.5 min at room temperature. DNAs were purified, digested in
vitro with 10 U of EcoRI and 2 µg RNase A, and then
resolved on a 1% agarose gel, blotted to Hybond N membranes (Amersham,
Arlington Heights, IL), and hybridized with a 32P-labeled
HindIII-EcoRI fragment corresponding to the
luciferase gene. As a control for deproteinated DNA, DNAs purified from
injected oocytes were digested with DNase I at a 100- to 200-fold lower
concentration than that used for oocyte samples.
MNase Digestion Assay in Xenopus Extracts
These assays were performed as described (23). Briefly, 20
injected oocytes were homogenized in digestion buffer as above. Equal
aliquots of the extracts (6.7 oocyte equivalents) were digested with 2,
6, and 30 U of MNase (Boehringer Mannheim, Indianapolis, IN) for 20 min
at room temperature. DNAs were purified and resolved on a 1.3% agarose
gel, blotted to Hybond N membranes, and hybridized with the indicated
probes.
MNase Digestion Assay with in Vitro Reconstituted
Nucleosomes
Chromatin assembly extracts were prepared from pre-blastoderm
stage Drosophila embryos as described (31, 41). The extracts
were incubated with 100 ng RARß2SK plasmid DNA and 650 ng FX174 DNA
for 6 h at 26 C. One hundred to 200 ng of purified recombinant
RXR/RAR heterodimers (see above) were added to the reaction at the
indicated times and incubated for 30 min at 26 C. MNase digestion was
performed with 25 U. DNAs were then purified, resolved on a 1.3%
agarose gel, and hybridized with indicated probes.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. Ben-Zion Levi, Nicoletta Landsberger,
Toshio Tsukiyama, Carl Wu, Igor Dawid, Giuseppe Martini, and Hong Chun
for valuable reagents and advice and Robin Kahn for technical
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Keiko Ozato, Laboratory of Molecular Growth Regulation, Building 6, Room 2A01, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753.
1 Present address: European Institute of Oncology, Department of
Experimental Oncology, 20141, Milan Italy. 
Received for publication October 3, 1997.
Revision received November 20, 1997.
Accepted for publication December 9, 1997.
 |
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