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(Received for publication, June 12, 1996, and in revised form, June 13, 1997)
From the ABSTRACT We have studied the transcriptional behavior of
the mouse mammary tumor virus long repeat (MMTV-LTR) promoter during a
prolonged exposure to glucocorticoids. When integrated into XC-derived
cells, MMTV-LTR expression reached its maximum during the first day of dexamethasone treatment, but longer exposure to the hormone resulted in
the deactivation of the promoter. In contrast,
glucocorticoid-responsive resident genes or MMTV-based transiently
transfected plasmids maintained or even increased their mRNA levels
during the same period of hormone treatment. An integrated chimeric
construct containing the hormone-responsive elements from MMTV-LTR but
in different sequence context became also deactivated after a prolonged hormone treatment but with a deactivation kinetics significantly slower
than constructs containing the entire, chromatin-positioning MMTV-LTR
sequence. The decrease on MMTV-LTR-driven transcription was concomitant
with a parallel closure of the MMTV-LTR chromatin and with a decrease
in glucocorticoid receptor (GR) concentration in the cell. We concluded
that the chromatin-organized MMTV-LTR promoter is particularly
sensitive to any decrease on GR levels. We propose that chromatin
structure may contribute decisively to the differential expression of
MMTV-LTR by two mechanisms: limiting MMTV-LTR accessibility to
activating transcription factors and accelerating its shutting down
upon a decrease on GR levels.
INTRODUCTION The mouse mammary tumor virus long terminal repeat (MMTV-LTR1) has
proved to be one of the most fruitful models for steroid hormone-regulated promoters since the beginning of the 1980s, when
several groups defined the concept of hormone-responsive element
(HRE).1 The MMTV-LTR promoter
can be activated by glucocorticoids and progestins through the
interaction of the hormone-receptor complex with HREs. The MMTV-LTR
HREs are termed GRE/PREs (glucocorticoid/progestin response elements)
because they bind either glucocorticoid or progestin receptors (GR or
PR). GRE/PREs contain two TGTTCT motifs, separated by three nucleotides
and arranged in a palindromic structure. In the MMTV-LTR, hormone
inducibility depends on the occurrence of one palindromic GRE/PRE and
three hemi-palindromes. These GRE/PREs are necessary and sufficient to
confer hormone inducibility to homologous and heterologous promoters
when placed in their vicinity (1, 2). Each of these GREs is recognized
and bound by a homodimeric hormone-loaded GR or PR, subsequently
triggering or enhancing transcription. Two other steroid hormone
receptors, the androgen receptor and the mineralocorticoid receptor,
have been found to bind to the same GREs and subsequently activate MMTV
promoter (see Ref. 2 for a recent review). GRE sequences are found in
regulatory regions of many glucocorticoid- and progestin-responsive genes, like the chicken or the frog ovoalbumin gene, the chicken lysozyme gene, the rat tyrosine aminotransferase gene, or
metallothionein (MT) genes from different mammalian tissues (1).
An unsuspected twist was given to the study of MMTV with the discovery
of very specific positioning of nucleosomes over a long stretch of the
MMTV-LTR sequence (3). One of these positioned nucleosomes, termed
nucleosome B, covers the four GREs present in the MMTV-LTR; it
coincides in position with a DNase I-hypersensitive site that appears
upon hormone induction (3, 4). Nucleosome B reconstituted in
vitro allows binding of both GR and PR while excluding other
transcription factors that also bind to the same DNA sequence when free
from histones (5-7). One possible role of the steroid hormone
receptors may be the removal or structural alteration of nucleosome B
to allow transcription factor binding and therefore enhance
transcription (6-9).
One of the most compelling arguments in favor of the role of chromatin
structure in transcriptional activation is the different behavior of
transiently transfected hormone-regulated promoters when compared with
their chromosomal counterparts (10). Recent studies show that a proper
chromatin structure is necessary for the correct expression pattern of
the MMTV-LTR promoter both in mammalian (11) and yeast cells (12). In
the case we present here, MMTV-based expression constructs transfected
into XC cells showed a loss of hormone response upon continuous
treatment with hormone. This deactivation was not observed in resident
hormone-responsive genes or when the same MMTV constructs were assayed
in transient transfection experiments. Although this effect could be
due to the presence of a specific repressor in hormone-treated cells, our data favor a mechanism in which deactivation correlates with an
exhaustion of GR receptor upon continuous hormone administration. We
propose that the response of the different genes and reporters to the
decline of the receptor may be related to the ability of each promoter
to shut itself down when receptor levels decay under a certain minimum.
This mechanism is likely affected by the chromatin structure over the
relevant sequences of the promoter. We also propose that such a
mechanism may be important for the differential expression of the
different hormone-regulated genes in the cell.
EXPERIMENTAL PROCEDURES Plasmids and Cell Lines
pMS-H5 (13), pAGE5MMTVLu (14), pMMTV-CAT (15), and
pTATT-CAT (16) have been previously described; a brief description is
given in the text when necessary. pRSVLUC is a construct where the Rous
sarcoma virus promoter (17) directs the expression of luciferase.
pTATT-AGE5Lu is a derivative of pAGE5MMTVLu where the
Sma/PvuII fragment was replaced by the
HindIII fragment of pTATT-CAT treated with DNA polymerase I
(Klenow fragment). This construct has the same characteristics of
pAGE5MMTVLu, except that the luciferase gene is driven by the thymidine
kinase promoter and made hormone-responsive by two copies of a
synthetic, pseudopalindromic GRE. pMS-HSP27 is an unpublished construct
identical to construct pMS-H5, except that the histone H5 sequences
were replaced by hamster hsp27 coding sequences; it was a
generous gift from Prof. J. Landry (Université Laval,
Québec, Canada).
Two cell lines derived from epithelial-like XC
rat sarcoma cells were used. XC-10 cells (13) contain about 300 copies
of the plasmid pMS-H5 (a contribution from Prof. A. Ruiz-Carrillo, Laval Université, Québec, Canada). XC-14 cells contain a
single integrated copy of
pMS-HSP27.2 XC-8 is a
nontransfected XC clone used as a control (13). Cell lines H12 and B11
were generated from stable transfection of XC-8 cells with plasmids
pAGE5MMTVLu and pTATT-AGE5Lu, respectively (see below).
Cell Culture and Transfections
All cell lines were grown in Dulbecco's
modified Eagle's medium containing 1 mg/ml glucose, 100 IU/ml
penicillin, 100 µg/ml streptomycin and supplemented with 2 mM L-glutamine and 10% (v/v) of
heat-inactivated fetal calf serum. Cultures were maintained at 37 °C
in a humidified atmosphere containing 5% CO2. Except for
the nontransfected XC-8 cells, stock cultures were maintained in the
presence of 350 µg/ml G418 (Sigma); cells split out for experiments
were cultured without antibiotic.
XC-8 cells were
transiently transfected by the DEAE-dextran method (18, 19). One
million cells were incubated at room temperature for 30 min with a
mixture containing 5 µg of plasmid DNA and 0.25 mg DEAE-dextran in
0.5 ml of 1 × TBS (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM
CaCl2, 0.5 mM MgCl2). After incubation, the cells were treated for 2 min at room temperature with
culture medium containing 15% Me2SO, washed in TBS,
further incubated for 2.5-3 h at 37 °C, 5% CO2 in
medium containing 0.1 mM chloroquine diphosphate, and
finally returned to standard culture medium. Hormone was added to the
medium 48 h after transfection, and incubation was continued for
3-4 days before harvesting the cells to assay luciferase and
chloramphenicol acetyltransferase (CAT) activity. All transfections and
treatments were performed in duplicate.
XC-8 derivatives
containing the MMTV-luciferase construct were obtained by calcium
phosphate precipitation (20) with 30 µg of the plasmids pAGE5MMTVLu
or pTATT-AGE5Lu per 9-cm plate (approximately 2.5 million cells). For
the selection of transfected cells, the medium was supplemented with
350 µg/ml G418. Stable G418-resistant clone pools were seeded at
limit dilution, and several single clones were isolated and
characterized.
Total RNA Extraction and Northern Blot Analysis
Total RNA was extracted by the acid guanidinium
thiocyanate-phenol-chloroform method (21). 10-20 µg of RNA was
separated by electrophoresis through 1% agarose in MOPS buffer (20 mM MOPS, 1 mM EDTA, 1 mM sodium
acetate, pH 7.0) in the presence of 2.2 M formaldehyde and
transferred to a nylon membrane (Hybond-N, Amersham Corp.) in 10 × SSC (0.15 M NaCl, 15 mM sodium citrate, pH
7.5). Filters were hybridized by standard methods using either a
randomly primed 32P-labeled DNA probe or a
32P-labeled 5 Primer Extension Analysis
Total RNA (20 µg) was hybridized in 50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl2, 10 mM dithiothreitol with 0.2 pmol of
32P-labeled 5 CAT and Luciferase Assays
Cell monolayers were rinsed twice in phosphate-buffered saline
and lysed by the addition of 0.6 ml of lysis buffer (25 mM Glycyl-glycine, pH 7.8, 1 mM EDTA, 1 mM
dithiothreitol, 1% Triton X-100, 15% glycerol). The lysate was
transferred to a microfuge tube and centrifuged for 3 min. 50 µl of
the supernatants was added to 0.35 ml of luciferase assay buffer (25 mM glycylglycine, pH 7.8, 15 mM
MgSO4, 5 mM ATP). Luciferase-mediated light
output was determined on a Lumat LB 9501 luminometer (Berthold) by
injection of 100 µl of 1 mM luciferin and integration of
the peak of light emission for 30 s. CAT assays were performed
with a CAT-enzyme-linked immunosorbent assay kit (Boehringer Mannheim),
following the manufacturer's instructions. Protein concentrations of
extracts were determined according to Bradford using a commercial kit
(Bio-Rad).
DNase I Hypersensitivity Assay
Approximately 200 million cells of the appropriate cell lines
were incubated with 1 mM dexamethasone for the indicated
periods of time and scraped into phosphate-buffered saline, and the
nuclei were isolated as described previously (23). The reaction was initiated by the addition of DNase I (Promega, 1 units/ml) and the
tubes were incubated at 30 °C. After 10 min the reaction was stopped
by the addition of 5 volumes of stop solution (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.5 mg/ml proteinase K, 1%
N-lauryl-sarcosine solution) and incubated for 3-16 h at
37 °C. DNA was purified by two phenol/chloroform/isoamyl alcohol
extractions and two chloroform extractions, ethanol precipitated, and
resuspended in TE (10 mM Tris, pH 7.4, 1 mM
EDTA) buffer. For Southern blot analysis, 40 µg of DNA from each
sample was digested to completion with the appropriate restriction
enzymes and separated electrophoretically through 0.8% agarose gels in
TEA buffer (40 mM Tris acetate, 1 mM EDTA). DNA
was transferred to a nylon membrane (Hybond N, Amersham Corp.) in
20 × SSC and cross-linked by UV treatment (Stratalinker, Stratagene). Membranes were hybridized with randomly primed
32P-labeled DNA probes either from a
HindIII/XhoI fragment from construct pMS-HSP27
encompassing the MMTV-LTR sequences (XC-14 cells) or from a
AvaI/EcoRI fragment from the luciferase gene (H12
cells).
GR Analysis by Western Blot
Western blots for the GR were performed according to Ref. 24.
XC-14 or H12 cells were treated with 1 µM Dex for 0, 1, and 4 days. At each time, cell monolayers were washed with
phosphate-buffered saline and suspended in hypotonic buffer (10 mM Hepes, pH 7.9, at 4 °C, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol). The
swollen cells were homogenized with a Dounce pestle B (20 strokes) and
centrifuged. Pelleted nuclei were resuspended in 100 µl of 20 mM Hepes, pH 7.9, at 4 °C, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol. Nuclear proteins were extracted by
adding 100 µl of the same buffer containing 0.45 M KCl.
The S-100 fraction was prepared by adding 0.11 volumes of 10 × cytoplasmic extract buffer (0.3 M Hepes, pH 7.9, at
4 °C, 1.4 M KCl, 30 mM MgCl2) to
the cytoplasmic fraction and centrifugation at 100,000 × g for 1 h. The protein concentration in the extracts
was determined by the Bradford assay (Bio-Rad). 70 µg of protein from
each extract was separated on an 8% SDS-polyacrylamide gel and
transferred onto a nitrocellulose membrane. The membrane was blocked
with 5% nonfat dry milk in 50 mM Tris, pH 7.6, 0.2 M NaCl, 0.05% Tween 20 and incubated with a rat liver GR
polyclonal antibody (kindly provided by Pr. O. Wränge, Uppsala,
Sweden) at 1:200 dilution for 2 h at room temperature. The
membrane was washed extensively and incubated with a horseradish
peroxidase-conjugated rabbit anti IgG (Promega) at 1:10,000 dilution
for 1 h. The membrane was washed again and GR was detected with
the Amersham Corp. ECL Western blotting Detection kit. Relative
quantitation of the bands was performed using a transmittance scanning
densitometer (Molecular Dynamics).
RESULTS Integrated
constructs containing MMTV driving either the chicken histone H5 or the
hamster hsp27 genes showed a tight transcriptional regulation by glucocorticoids, with no detectable basal expression without hormone (Fig. 1, A and
B,
XC-10 and XC-14 cells incubated with Dex for several days showed a
continuous fading of transcripts from both histone H5 and hamster
hsp27, respectively. By day 5, levels of both transcripts were reduced to 10-20% of their maximal value (Fig. 1, A
and B). In contrast, To demonstrate that the presence of the hormone-receptor complex was
required not only to start transcription but also to maintain the
transcription of the Dex-responsive genes, we analyzed the behavior of
the different activated mRNAs upon Dex removal. Removal of Dex
24 h after its administration resulted in a relatively rapid decay
of the mRNAs from all analyzed GR-activated genes. Transcription of
both the MMTV-derivatives and the resident MT-I returned to basal
levels by 12 h after Dex removal, whereas the We ensured the correct initiation of the transcripts over the period
examined by primer extension analysis of MMTV-driven hamster
hsp27 transcript in XC-14 cells was performed. As seen in
Fig. 2A, the amount of correctly initiated transcript
increased after hormone administration and decreased upon continuous
treatment closely following the pattern of total mRNA decay
observed in Northern blots. Fig. 2B shows a quantitation of
the correctly initiated MMTV-driven hamster hsp27 compared
with the amount of total mRNA as observed in the blots of Fig. 1.
The decrease of the correctly initiated hamster hsp27
transcript (line) closely followed the disappearance of the
total hsp27 mRNA in XC-14 cells or H5 mRNA in XC-10
cells (bars). On the contrary, neither MT-I nor the
We produced a series of
stable cell lines derived from the parental XC-8 cells containing the
hormone-responsive, luciferase reporter plasmids pAGE5MMTVLu and
pTATT-AGE5Lu. pAGE5MMTVLu contains a 1.4-kb fragment from MMTV-LTR that
includes the complete set of HREs. pTATT-AGE5Lu contains two
copies of a pseudo-palindromic HRE (5 Our results showed clear differences among the deactivation kinetics of
the two integrated reporters. As seen in Fig.
3, the luciferase activity was strongly
induced in H12 cells 8 h after Dex administration (about 40-fold),
but it faded upon continuous hormone treatment. Although not identical,
this deactivation kinetics resembled the integrated MMTV constructs in
XC-10 or XC-14 cells; by day 4, luciferase activity was only 20-25%
of its value at day 1. B11 cells showed clearly lower activation upon
hormone administration (4-5-fold), but although some decrease was
observed, the luciferase activity remained at 80% of its maximal value
until day 2, decreasing slowly thereafter. Therefore, the response of the pTATT-AGE5Lu construct in B11 cells to the continuous presence of
Dex was clearly different from the very closely related pAGE5MMTVLu construct in H12 cells. pAGE5MMTVLu has been reported to show the
typical MMTV nucleosome positioning over its 1.4 kb of the MMTV-LTR
sequences when integrated in the genome (9). On the contrary, plasmid
pTATT-AGE5Lu contains no MMTV sequences except for two
pseudopalindromic HREs, identical in sequence to the distal HRE of the
MMTV-LTR.
Plasmids transiently transfected
by the dextran-sulfate method are kept in the receiving cells for
several days (31). As seen in Fig.
4A, a transiently transfected
construct based on the Rous sarcoma virus promoter showed a robust
transcriptional activity for 5 or 6 days after transfection. However,
the amount of transcription decreased very rapidly during the first 2 or 3 days, stabilizing afterward. Between days 3 and 6 after
transfection there is only a moderate loss of transcription, on a rate
of about 15% decrease per day. We have used dextran-sulfate
transiently transfected plasmids in this time window to study the
effects of continuous administration of hormone on their transcription. In addition to the relative stability of transfected DNA, the use of
this time window has the advantage that by day 3 the amount of total
DNA in the cells has decreased considerably (31), reducing the effects
of the presence of an excess of GR binding sites into the cells.
We transiently transfected XC8 parental cells with two different
hormone-responsive promoters, and we analyzed the response to
continuous Dex administration. pMMTV-CAT and pTATT-CAT have identical
responsive elements, pAGE5MMTVLu and pTATT-AGE5Lu, respectively, but drive the CAT gene instead of the luciferase gene. As seen in Fig.
4B, both plasmids produced a continuous increase in the levels of CAT protein through all the examined period, up to 4 days of
continuous hormone administration. Removal of the hormone after 2 days
of induction resulted on a steady decrease on the levels of CAT
protein; control cells continuously exposed to Dex showed a further
increase of CAT levels during the next 2 days (Fig. 4C).
From these results, we concluded that the increasing levels of CAT
protein upon the prolonged hormone exposure reflected a continuous
transcription of the gene all over the studied period. This behavior
was more similar to the response of the resident In addition to a difference in transcription levels, a possible
explanation for the differences we observed between the stable and
transiently transfected constructs could come from protein or mRNA
stability. To check these possibilities, we transiently transfected XC8
cells with pMS-HSP27, the same construct used to generate XC-14 cells.
Fig. 4D shows the levels of hamster hsp27 mRNA from this construct analyzed by Southern blot after 1 and 4 days of continuous hormone administration. The data showed an accumulation, rather than a decrease, of the transcript from
transiently transfected cells from day 1 to day 4. This is a sharp
contrast with the behavior of the very same construct integrated in
XC-14 cells, where it fades after day 1 of hormone administration (see Fig. 1B, top panel). Thus, whatever the mechanism
responsible for the decay of chromosome-integrated MMTV-driven
transcription upon continued hormone administration might be, it seems
not to apply to MMTV constructs transiently transfected into XC
cells.
MMTV-LTR chromatin displays an array of very
precisely positioned nucleosomes over at least 1.2 kb (3). Upon hormone
administration, a DNase I-hypersensitive site appears between positions
A clear DNase I-hypersensitive site appeared upon Dex administration in
H12 cells (Fig. 5A), mapping,
as expected, at nucleosome B (3). Upon continued hormone treatment, the
hypersensitive site became clearly weaker. Fig. 5B shows a
quantitative analysis of the appearance and fading of the
hypersensitive site in H12 cells. Maximal DNase I cutting was observed
1-4 h after Dex administration, decreasing steadily from then on (30%
of maximal cutting at 24 h and 10% of maximal cutting at 96 h; Fig. 5B, bars). The appearance and fading of
the DNase I-hypersensitive site preceded by several hours the
activation and posterior fading of luciferase transcription (Fig.
5B). Comparable results were obtained in XC-14 cells (not shown). In this case, the partial fading of the hypersensitive site on
nucleosome B coincided temporally with the decrease of MMTV-driven
hamster hsp27 transcription. We interpret the reduction in
hypersensitivity as a refolding of nucleosome B. This nucleosome seems
to be necessary and sufficient to avoid binding of key transcription factors to the MMTV-LTR promoter (2, 6, 7, 11, 12); therefore, its
refolding should result in the shutting down of the promoter.
GR
expression is down-regulated by the GR-hormone complex (32, 33). Our
results are consistent with a loss of GR upon continuous hormone
exposure, resulting in a simultaneous decay in transcriptional
activation and a loss of the structural changes on chromatin occurring
upon GR binding. To investigate this hypothesis, we have followed the
GR distribution in XC-14 and in H12 cells upon continuous treatment
with Dex. The results of a Western blotting of nuclear and
cytoplasmatic extracts of both cell lines are presented in Fig.
6. In both cases, the vast majority of GR
was present in the cytosol in untreated cells, being readily
translocated into the nucleus shortly after Dex administration. After 4 days of continuous hormone administration, GR was located both in the nucleus and in the cytosol, and its amount was reduced by 70-80%. A
quantitative analysis of these changes is shown in Fig.
6B.
The similar behavior of the levels of GR (Fig. 6B), the
intensity of the DNase I-hypersensitive site (Fig. 5B), and
the integrated MMTV-LTR transcription rate (Fig. 2B) as a
response to a prolonged Dex administration is noteworthy. This
similarity is lost in the case of transiently transfected reporters or
of resident, GR-responsive genes (Fig. 2B). We concluded
that the MMTV-LTR transcription rates followed the actual levels of GR
more closely than other genes or reporters analyzed. This may indicate
that different promoters could respond differently to the loss of GR,
suggesting that the amount of hormone-loaded GR required to sustain
hormone-activated transcription may vary from one gene to another.
DISCUSSION We have analyzed the behavior of different
glucocorticoid-responsive promoters after up to 5 days of continuous
presence of hormone. We found a clear response to glucocorticoids for
different MMTV-LTR-derived constructs as well as for two resident
genes, the rat MT-I gene and an mRNA we identified as corresponding
to the The results presented here showed a deactivation of MMTV-driven
promoters upon continued hormone administration in XC-derived cell
lines. This deactivation did not affect either resident
glucocorticoid-responsive genes or transiently transfected reporters.
This phenomenon may be related to the reported transient response to
Dex of MMTV constructs on BPV-based episomes, whereas transiently
transfected plasmids remained actively transcribed (10). We propose
that the differential kinetics of activation and deactivation of
different hormone-responsive genes may be a general phenomenon, and we
believe that this should have important physiological implications.
Our data showed that the deactivation kinetics of the MMTV promoter did
not depend on the transcription product. Transcripts of three very
different genes, such as luciferase, hamster hsp27, and
chicken histone H5, faded at the same rate. Furthermore, we have
observed a similar specific MMTV deactivation in completely different
cell lines.4 In addition, the
deactivation of the MMTV promoters was independent from the cell cycle,
because XC-10 cells stop dividing upon Dex administration, whereas
XC-14 and XC-8 cells keep on dividing at the normal rate (13). In turn,
deactivation of the transcription of a MMTV-driven reporter depended on
whether the construct was integrated into the host cell genome,
suggesting a critical role of MMTV-chromatin in the process. This model
is reinforced by the differential kinetics of deactivation observed on
H12 and B11 cells. These two cell lines contain integrated reporters
very similar in structure, except for their hormone-responsive regions. pAGE5MMTVLu (H12 cells) contains a long fragment from MMTV that has
been shown to promote nucleosome positioning (9). pTATT-AGE5Lu (B11
cells) contains only the very same HRE found in the MMTV-LTR. In this
case, the fact that the transcription from the former construct
decreased much faster than that from the latter can neither be
explained by differential stability of the gene products nor by
differences on the HRE sequences.
Chromatin structural rearrangements of the MMTV-LTR upon glucocorticoid
induction are revealed by the appearance of a DNase I-hypersensitive
site (3, 4, 9). We have observed the appearance of this hypersensitive
site upon Dex administration in our integrated MMTV-LTR constructs.
Furthermore, the decline of MMTV-driven transcription upon continued
hormone administration temporally coincided with a proportional loss of
this hormone-induced DNase I-hypersensitive site. Our results showed a
temporal relationship between the intensity of the DNase
I-hypersensitive site and the transcriptional activity of the reporter
gene in both XC-14 and H12 cells. We observed a 4-8-h lag period
between the formation of the hypersensitive site and the peak of
luciferase activity in H12 cells. We interpret this delay as the time
required for the synthesis of luciferase. It is generally accepted that
nucleosome B prevents key transcription factors from binding to their
cognate sequences onto the MMTV-LTR (5-7). We propose that the
observed fading of the DNase I-hypersensitive site corresponded to the refolding of nucleosome B. Under this point of view, the decay on MMTV
transcription would be explained by the inability of the GR to maintain
chromatin in an open state during long periods of exposure to the
hormone.
Our results showed that transcription driven by integrated
chromatin-organized MMTV-LTR displayed an exquisite dependence on the
actual levels of hormone-loaded receptor. A loss of response to
hormones upon continuous administration has been associated with a
down-regulation of the receptor in different cell systems (32-35).
This down-regulation has been related both to a transcriptional repression of the GR gene and to a destabilization of GR mRNA by
the hormone-loaded GR (32, 33). Our data showed that GR was mainly
cytoplasmic in untreated XC cells, entering the nucleus shortly after
hormone administration. After 4 days of continuous hormone
administration, GR levels decreased significantly to 20-30% of the
initial levels. This decrease correlated with the decay in MMTV-driven
transcription but opened the question of how other glucocorticoid-responsive genes maintained their transcriptional levels
high, despite the decrease in GR levels.
Several models can account for the observed specificity of MMTV-LTR
deactivation. One possibility is the presence of a specific repressor
affecting MMTV-LTR but not MT-I or It is likely that this type of chromatin-based regulatory
mechanism applies to many hormone-responsive genes. Specific
nucleosome positioning has been shown for different hormone-regulated
genes (38-40). It is conceivable that chromatin organization, among
other factors, may determine whether a given gene responds to a given amount of hormone in a given cell type. It may also determine what
concentrations of hormone-loaded receptor are required for maintaining
or for shutting down transcription. As chromatin structure changes in
many different ways depending on the cell differentiation stage and
cell metabolism, this may represent a finely tuned mechanism of
modulating hormone response of many different genes.
FOOTNOTES ACKNOWLEDGEMENTS We are indebted to Prof. Adolfo Ruiz-Carrillo
(Université Laval, Québec, Canada) for contributions in the
development of this project and for the gift of different cell lines
and plasmids. We also thank very much Prof. J. Landry (Université
Laval, Québec, Canada) for the gift of some key cell lines and
plasmids, some of them unpublished. We thank Dr. Mathias Truss and
Prof. Miguel Beato (Institut für Molekular biologie und Tumor
forschung, Marburg, Germany) for advice and for the gift of the
pAGE-Luc plasmids. We thank also to Dr. O. Wränge (Uppsala,
Sweden) for the gift of the antibody against the GR. Thanks also to Dr.
Montserrat Bach (Centre d' Investigació i Desenvolupament,
Consejo Superior de Investigaciones Científicas, Barcelona,
Spain) for support in the development of this work.
REFERENCES
Volume 272, Number 35,
Issue of August 29, 1997
pp. 21803-21810
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
§,
Departament de Biologia Molecular i
Cel.lular, Centre d'Investigació i Desenvolupament, Consejo
Superior de Investigaciones Científicas, C/Jordi Girona, 18-26, 08034 Barcelona, Spain and the ¶ Laboratoire de Biologie
Moléculaire des Eucaryotes, CNRS UPR 9006, 118 route de Narbonne,
31062 Toulouse Cedex, France
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
end-labeled oligonucleotide. The probes
were: hamster hsp27, a pMS-hsp27
NheI-XhoI fragment containing the coding sequence of hamster hsp27; chicken histone H5, a pMS-H5
NheI-XhoI fragment containing the coding sequence
of H5; actin, an EcoRI-HindIII fragment from
plasmid pAC18.1 (22) that encompasses the rat 
-actin; rat
metallothionein-I, 5-TGCAGGAGGTGCATTTGCAGTTCTTGCAGC-3 (positions
138-168 of the mRNA); and rat 
B-crystallin,
5-CGCTGATGGGAAACTTCCTTGTC-3 (positions 600-623 of the mRNA).
Relative quantitation of the bands was performed using either a
Bio-Imaging Analyzer BSA 1000 (Fujix) or a transmittance scanning
densitometer (Molecular Dynamics).
end-labeled primer for 4 h at 37 °C.
The primer was extended in this reaction mixture after the addition of
0.5 mM of each deoxyribonucleotide, 50 ng/ml of actinomycin
D, 25 ng/ml of bovine serum albumin, and 200 units of MMTV-reverse
transcriptase (Life Technologies, Inc.) for 1 h at 37 °C. The
reaction was stopped by the addition of 12.5 mM EDTA and 25 ng/ml of pancreatic RNase A. Samples were extracted once with
phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated and
analyzed on 8% acrylamide-urea sequencing gels. The primers used were:
rat actin, 5-GGGACGGAGGAGCTGCAAG-3, and hamster hsp27,
5-GCAGCAGCGAGAAGGGCAC-3. The 5 end of the rat actin probe is 55 bases downstream from the rat actin mRNA start site; the hamster
hsp27 probe is 321 bases downstream from the origin of
transcription driven by MMTV in construct pMS-HSP27. Relative
quantitation of the bands was performed using a transmittance scanning
densitometer (Molecular Dynamics).
DEX lines). Administration of the synthetic
glucocorticoid Dex resulted in a dramatic induction of the chicken
histone H5 mRNA in XC-10 cells (Fig. 1A, top
panel) and of the hamster hsp27 mRNA in XC-14 cells
(Fig. 1B, top panel). In addition, Northern blot
analysis of hormone-induced and noninduced XC-14 cells probed with the
hamster hsp27 cDNA revealed an endogenous mRNA that
was also increased by glucocorticoids (Fig. 1B, band labeled X). We tentatively identified this transcript as the
B-crystallin mRNA, based on its size of less than 1 kb (25), its
glucocorticoid responsiveness (26), its cross-hybridization with the
hamster hsp27 cDNA, and the epithelial-like character of
XC cells. This was confirmed by rehybridizing the same blots with an
B-crystallin-specific oligonucleotide complementary to the 3 end of
the rat transcript (see "Experimental Procedures"). As shown in
Fig. 1B (second panel), this probe revealed a
clear increase in the amount of the B-crystallin transcript after
hormone administration. We have also detected Dex induction of the rat
metallothionein-I gene (MT-I), which is a well known
glucocorticoid-responsive gene (27-30). As shown in Fig.
2B, its mRNA levels
increased two or three times after Dex administration in the XC-14
cells (Fig. 1A, third panel, and Fig.
2B). Simultaneous addition of Dex and cycloheximide did not prevent hormonal activation of the B-crystallin or MT-I genes (not
shown), indicating that their response to Dex did not require protein
synthesis.
Fig. 1.
Expression of different hormone-responsive
genes during continuous hormone administration. XC-10
(A) and XC-14 (B) cells were treated with 1 µM Dex for the indicated period of time. Total RNA was
extracted, separated by gel electrophoresis, blotted onto membranes,
and hybridized with the indicated probes (see "Experimental
Procedures"). Bands corresponding to the different mRNAs are labeled in the right margin. The
band marked X on the hamster
hsp27-hybridized blot is made of several different, though related, mRNAs, probably including the rat hsp27
mRNA besides the B-crystallin mRNA. C, XC-14
cells were first treated with 1 µM Dex for 24 h, and
then the hormone was either removed (closed circles) or
allowed to act (control cells, open squares) for a further
12 or 24 h. After these periods of time, total RNA was extracted
and hamster hsp27 (right panel), B-crystallin
(middle panel), and MT-I (left panel) mRNAs
were quantified on Northern blots. RNA amounts are given relative to
the value at the time the hormone was removed (time 0).
[View Larger Version of this Image (35K GIF file)]
Fig. 2.
Analysis of correctly initiated hamster
hsp27 mRNA expression after hormone administration.
A, primer extension analysis of the hamster hsp27
(upper panel) and actin (lower panel) mRNAs from XC-14 cells were performed after continuous presence of 1 µM Dex for the indicated times. B,
quantitative analysis of Northern blots from Fig. 1 (bars)
and primer extension from A (line, open circles). Ordinates represent relative amount of each transcript taking as 1 the value at day 1. Hatched bars, total hamster
hsp27 mRNA in XC-14 cells. Black bars, H5
total mRNA in XC-10 cells. Shaded bars, MT-I total
mRNA (XC-10 and XC-14 cells). White bars, B-crystallin total mRNA (XC-10 and XC-14 cells). Error
bars represent standard deviation from duplicates of each cell
line.
[View Larger Version of this Image (41K GIF file)]
B-crystallin mRNA levels did not
fade after at least 5 days of continuous hormone administration (Fig.
1B, top and second panels). Likewise,
MT-I mRNA concentration showed an increase, rather than a decrease,
after 5 days of continuous hormone treatment (Figs. 1B and
2B). These results suggest a specific deactivation of the
MMTV promoter not affecting other GR-regulated genes. It is noteworthy
that the two MMTV-driven transcripts (histone H5 and hamster
hsp27) decayed at an essentially identical rate, despite the
different phenotypical effects of Dex administration on each cell line.
In the continuous presence of Dex, XC-14 cells continued to divide at
their normal rate (without Dex) of about one division every 18 h,
similar to the growth rate of the parental XC-8 cells. In contrast, Dex
induction of the expression of chicken histone H5 in XC-10 cells led to
the arrest of cell growth (not shown). This effect may be a consequence
of the incorporation of histone H5 into the host cell chromatin and the
subsequent changes on chromatin structure (13). XC-10 cells reinitiated
cell division on day 4 or 5, when histone H5 protein levels are down to
10-20% of the amount on day 1 (13).3 The behavior of MT-I
and B-crystallin mRNAs in XC-10 and XC-14 cells was essentially
the same; results from both cell lines are included on the data of Fig.
2B.
B-crystallin gene
decayed at a somewhat slower ratio (Fig. 1C). The apparent
mRNA half-lives of the different genes were relatively short: less
than 5 h for the hamster hsp27 mRNA, about 12 h for the B-crystallin mRNA, and less than 6 h for the MT-I
mRNA. These half-lives are clearly shorter than the time window we
are considering for our kinetics experiments (3 or 4 days); therefore,
it is likely that mRNA stability has only minor effects on the
rates of the observed decays for MT-I, B-crystallin, or hamster
hsp27 transcripts.
B-crystallin transcripts showed any decrease in their levels during
the examined period.
-GGTTACAAACTGTTCT-3),
which confer hormonal response to the adjacent thymidine kinase
promoter. These plasmids also include a neomycin resistance gene and a
scaffold-attachment region of the human interferon domain (14). The
presence of a scaffold-attachment region helps plasmids to integrate in
the cell genome mainly as single, nonrearranged copies, with very
little position dependence (9, 14). Two neomycin-resistant clones
showing clear glucocorticoid induction were selected for analysis of
their behavior upon continuous hormone treatment: H12 cells,
transfected with pAGE5MMTVLu, and B11 cells, transfected with
pTATT-AGE5Lu.
Fig. 3.
Activation/deactivation kinetics of
integrated constructs. Clones isolated after transfection of XC
cells with either pAGE5MMTVLu (H12 cells, black squares) or
pTATT-AGE5Lu (B11 cells, white circles) were treated for
different periods of time days with 1 µM Dex. Relative
luciferase activity of each point is expressed as 100 × (Ti To)/(Tmax To). In this equation, Ti
represents the luciferase activity at a given time of Dex treatment,
To represents the value corresponding to cells
maintained in culture for the same period of time without Dex, and
Tmax represents the luciferase activity obtained
after 8 h of Dex administration (the highest value
observed).
[View Larger Version of this Image (14K GIF file)]
Fig. 4.
Hormonal response of transiently transfected
plasmids upon prolonged hormone treatment. A, XC-8 cells
were transiently transfected with the pRSV-LUC plasmid, and the
luciferase activity (RLU/mg protein) was measured at different times
after transfection. B, pMMTV-CAT (dark shading)
and pTATT-CAT (light shading) were transiently transfected
into XC-14 cells, and the CAT amount was measured by enzyme-linked
immunosorbent assay 1 and 4 days after Dex administration. Ordinates
indicate the amount of CAT protein relative to noninduced cells (day
0). C, XC-14 cells transiently transfected with pMMTV-CAT
were treated with Dex for 2 days (black circles). After 2 days, Dex was removed from half of the plates, and the cells were
incubated further 2 days (white circles). Control cells were
continuously exposed to hormone (black circles). The ordinates indicate amount of CAT protein in arbitrary units.
D, XC-8 cells were transiently transfected with pMS-HSP27,
and hamster hsp27 mRNA was detected on a Northern blot
as described after 1 and 4 days of continuous hormone treatment. Lane
labeled as 0 corresponds to untreated cells. Given our
protocol of hormone treatment of transiently transfected cells, day 1 of hormone treatment (B-D) corresponds to day 3 after
transfection (A).
[View Larger Version of this Image (27K GIF file)]
B-crystallin and
MT-I genes than to the integrated MMTV-driven constructs.
200 and 100 relative to the transcription initiation site. This has
been explained as a destabilization of one of the positioned
nucleosomes, the so-called nucleosome B (3, 4, 9). This DNase
I-hypersensitive site seemed to parallel transcriptional activation;
therefore, we investigated its appearance on the integrated
MMTV-hsp27 construct during the process of induction and
deactivation.
Fig. 5.
Chromatin structural changes during MMTV
deactivation. A, nuclei isolated from H12 cells were treated
with 1 µM Dex for the indicated periods of time from 1 to
96 h and digested with the indicated amounts of DNase I. DNA was
purified and digested with AvaI, and the fragments resulting
from the DNase I digestion were characterized by Southern blot analysis
using the AvaI/EcoRI probe from the luciferase
gene as a probe. The AvaI digestion generates the 2.3-kb DNA
band labeled on the right. A DNase I-hypersensitive site,
mapping over nucleosome B (labeled DHS), appeared after only
1 h of Dex treatment, coinciding in position with nucleosome B
(see the diagram on the bottom). Quantification
of this DNase I-hypersensitive site throughout the Dex treatment is
shown in B. B, comparison of the kinetics of
appearance and fading of the DNase I-hypersensitive site
(bars) on the gel of A, measured by densitometry,
and the luciferase levels in H12 cells (line and circles) (data from Fig. 3).
[View Larger Version of this Image (36K GIF file)]
Fig. 6.
GR levels in XC-derived cells after
continuous hormone treatment. A, the amounts of GR in
nuclear extracts (left) and cytosol (right) were
analyzed by Western blot before (0) and after 1 and 4 days
of hormone treatment. Upper panel, XC-14 cells. Lower panel, H12 cells. The bands corresponding in size to the intact GR
are marked by arrows. Higher mobility bands very likely
correspond to degradation of the GR. B, densitometric
quantitation of the gel shown in A. Bars
represent the amount of GR (both intact and the proteolytic products)
found in the indicated fraction in untreated cells (black
bars) and in cells after 1 (hatched bars) and 4 (shaded bars) days of continuous Dex treatment.
[View Larger Version of this Image (26K GIF file)]
B-crystallin gene. To our knowledge, this is the first report of B-crystallin expression in XC cells.
B-crystallin promoters. We do not
favor this model, in part because it does not explain the temporal
correlation between the decay in GR concentration, the closing of the
MMTV-LTR chromatin, and the decrease in MMTV-LTR-driven transcription.
In addition, this model is difficult to reconcile with the maintenance
of transcription of transiently transfected MMTV-LTR plasmids. We
rather propose that the minimal amount of hormone-loaded GR required
for avoiding a shut down of transcription varies from one promoter to
another, depending on the chromatin structure over the GREs. In
integrated MMTV-LTR constructs, chromatin appears to refold rapidly
when the levels of occupied GR decrease, restraining the binding of
factors required for sustaining transcription. Therefore, these
promoters are especially sensitive to any decrease in hormone-loaded GR
levels. In contrast, transiently transfected plasmids seem to
accommodate some kind of loose chromatin package, which is very
accessible to enzymes or transcription factors (36). This probably
makes transiently transfected MMTV-LTR far less sensitive to the
decline of GR than their integrated counterparts. This may well also be
the case for the transfected HRE-thymidine kinase promoter on B11
cells, in principle devoid of nucleosome-positioning sequences. On the
other hand, both the rat MT-I (28-30) and the B-crystallin (25, 37)
promoters contain several different enhancers that respond to different
effectors. This probably results in a constitutive opening of the
chromatin structure. This is specially true for the rat MT-I gene,
which showed a high basal transcription in our culture conditions and
displayed a constitutive DNase I-hypersensitive site over its promoter
(30).
§
Partially supported by an Formación de Personal Investigador
fellowship from the Spanish Ministry of Education and Science and two
fellowships from the Generalitat de Catalunya.
To whom correspondence should be addressed.
1
The abbreviations used are: HRE,
hormone-responsive element; MMTV, mouse mammary tumor virus; LTR, long
terminal repeat; GR, glucocorticoid receptor; PR, progesterone
receptor; MT, metallothionein; Dex, dexamethasone; CAT, chloramphenicol
acetyltransferase; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase
pair(s).
2
J. Landry, unpublished data.
3
S. Boronat, H. Richard-Foy, and B. Piña,
unpublished results.
4
S. Boronat, H. Richard-Foy, and B. Piña,
manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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