Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Institut de Chimie B6, B-4000 Sart Tilman, Belgium
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ABSTRACT |
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In this report we show that thyroid hormone inhibits the expression of the hPRL gene in rat pituitary cells. Transient expression experiments show that thyroid hormone regulation involves a strong inhibitory element, located in the proximal (-164/-35) promoter, which is modulated by a more distal stimulatory response control region. Gel retardation experiments reveal that the thyroid hormone receptor does not bind to the proximal negative element.
We show the existence of an activating protein-1 (AP-1) response element located at positions -61 to -54 of the proximal promoter, conferring AP-1 stimulation to the hPRL promoter. This AP-1 induction is abolished when hormone-bound thyroid hormone receptor is present, indicating that there is an interference between the thyroid hormone receptor and AP-1 regulatory pathways.
Furthermore, using the complete hPRL upstream region, we show that estrogen induction is abolished by simultaneous thyroid hormone treatment.
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INTRODUCTION |
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Recently, thyroid hormone receptors have been shown to inhibit gene expression by interfering with the activity of the activating protein-1 (AP-1) transcription factor (15, 16, 17, 18, 19, 20, 21, 22). This factor is a dimer whose components are respectively encoded by the jun/fos protooncogene family (23). It binds to a specific DNA sequence (ATGAGTCA) and stimulates gene transcription in response to activation of the protein kinase C pathway (24). TRs exert their inhibitory effect through direct interaction with the AP-1 complex in solution, leading to the formation of a transcriptionally inactive TR/AP-1 complex. TRs do not compete with AP-1 for DNA binding sites (15).
PRL is a hormone essentially secreted by the anterior pituitary. Transcription of the human PRL (hPRL) gene in the pituitary is subject to tissue-specific and multihormonal regulation involving the POU domain transcription factor Pit-1 (25, 26, 27, 28). In the 5'-flanking sequence of the hPRL gene, two regulatory regions have been identified, a proximal promoter (-250/-35) containing three Pit-1-binding sites and a distal enhancer (-2000/-1200) with eight additional Pit-1-binding sites (29, 30). Regulation of hPRL gene transcription by phorbol esters, epidermal growth factor, TRH, Ca2+, and cAMP depends on elements located within the first 250 bp of the promoter (31, 32, 33, 34). Estrogens are reported to stimulate hPRL gene expression through a response element next to the distal enhancer (34). Nothing is known about T3 regulation of hPRL, whereas contradictory results have been obtained in GH pituitary tumor cell lines for rat PRL (rPRL) T3 regulation. T3 was found to inhibit rPRL promoter-driven transcription (35, 36, 37) in GH1 cells but to stimulate it in GH4C1 cells (38) through the same response element, located in the proximal promoter (coordinates -176/-11). From studies in GH3 cells, two TREs were identified in the rPRL gene: a positive response element in the distal enhancer next to the estrogen receptor response element and a negative response element in the 292-bp span of the proximal promoter (39).
This report focuses for the first time on the regulation of the hPRL gene by thyroid hormone. We show that the hPRL gene promoter contains both a positive and a negative TRE. The negative effect is stronger than the positive one and involves cross-talk between TR and AP-1.
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RESULTS |
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The Inhibitory Effect Is not Pit-1 Dependent
The first 250 bp of the hPRL promoter (proximal promoter) include
three well known Pit-1-binding sites (30). They also include the A site
involved in the responses to cAMP, TRH, and Ca2+ (32, 33)
(Fig. 2a). To study T3 regulation
in this proximal region, we tested two constructs: one bearing the
entire proximal region without the TATA box (bp -250/-35) and one
bearing the same fragment lacking the P3 Pit-1-binding site (bp
-164/-35). Both proximal promoter segments were cloned in front of
the TK promoter fused with the CAT reporter gene. The constructs were
introduced by electroporation into GH3B6 cells
(Fig. 2b
). As a positive control we used a construct containing three
palindromic TRE sequences cloned in front of the TK-CAT fusion. As
expected, T3 stimulates CAT expression from this
control plasmid approximately 3-fold while having no effect on the
negative control (pTKCAT), indicating that T3
effects are specific under our experimental conditions. CAT expression
from both p(-250/-35)TKCAT and p(-164/-35)TKCAT is inhibited about
3-fold by T3. This shows that the
T3 response element can mediate inhibition of the
heterologous TK promoter. Our experiments further show that the nTRE is
located between bp -164 and -35 of the hPRL proximal promoter.
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Involvement of Receptor-DNA Binding in the T3
Effects
The -164/-35 region contains two Pit-1-binding
sites (P1 and P2) and the cAMP response element A (Fig. 2a). A search
for potential TR-binding sites in this region revealed only one
imperfect half-site, GGGTaA (-113/-108). To test the affinity of TR
for this putative binding site, we performed gel retardation assays
with bacterially expressed TR (Fig. 3
). The probes used were an
oligonucleotide containing the -115/-87 PRL proximal promoter
sequence and, as a positive control, an oligonucleotide containing the
DR4 consensus TR-binding sequence (two TR consensus half-sites directly
repeated, spaced by 4 bp). As expected, we observed a retarded band
with the DR4 oligonucleotide, corresponding to the TR monomer (Fig. 3
, lane 2); this band disappeared in the presence of a
100-fold excess of cold DR4 oligonucleotide (lane 3), but not in the
presence of a 100-fold excess of Sp1 oligonucleotide (lane 5).
Moreover, the retarded band was supershifted in the presence of anti-TR
antibodies (lane 4). All of these results prove the specificity of the
retarded band. Addition of bacterially expressed RXR led to the
formation of TR/RXR heterodimers (lane 6), as expected.
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T3 Inhibition of the hPRL Proximal Promoter Is
Independent of the Forskolin Pathway, but Involves an AP-1-Binding
Site
To identify the indirect T3 repression
mechanism, two alternative pathways were investigated. TR has been
shown to interfere with stimulation of the PIT1 gene promoter by
forskolin (40). As the proximal hPRL promoter contains cAMP response
elements (-164/-35) (32, 33), we examined whether
T3 treatment might affect the response to
forskolin mediated by this region. GH3B6 cells
were transfected with a construct containing the first 164 bp of the
proximal promoter cloned in front of the luciferase (LUC) reporter gene
(p164PRL-LUC). The cells were treated with forskolin and/or
T3. Gene expression increased significantly
(15-fold) in response to forskolin. T3 treatment
reduced the basal promoter activity approximately 2-fold. When both
treatments were applied, forskolin stimulation was reduced by a factor
of 2, clearly indicating that forskolin and thyroid hormone act through
distinct pathways (data not shown).
In search of an alternative pathway that might be modulated by
T3, sequence analysis of the hPRL proximal
promoter was performed. This analysis revealed the presence of a highly
conserved AP-1 consensus motif (-61 TGAaTCAT -54) containing only one
mismatch (see also Caccavelli, L., I. Manfroid, J. A. Martial, and M.
Muller, in preparation). This led us to investigate whether this
putative AP-1 site is functionally active and how destruction of this
site affects the response of the hPRL proximal promoter to
T3. To this end, we produced a mutation in the
AP-1 consensus motif of p164PRL-LUC, yielding plasmid
p164(AP-1)mPRL-LUC. We then transfected GH3B6
cells with either p164(AP-1)mPRL-LUC, its wild-type parent, or the
control plasmid pTKLUC bearing the LUC gene under the control of the TK
promoter. The cells were treated or not with T3.
The results are shown in Fig. 4a.
T3 treatment did not affect expression from
pTK-LUC, but it inhibited expression from p164PRL-LUC (3-fold
inhibition). The mutant construct p164(AP-1)mPRL-LUC displayed 2-fold
lower basal expression and did not respond to
T3.
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T3 Abolishes AP-1 Activation of the hPRL Promoter in
Heterologous Cells
Since T3 inhibition appears to involve
a putative AP-1-binding site, we next analyzed the action of AP-1 on
the hPRL proximal promoter in nonpituitary cells. We cotransfected HeLa
cells with p164PRL-LUC in combination with vectors expressing
c-jun and c-fos (pRSVc-jun and
pRSVc-fos) and/or with pSV2c-erbA. It should
be mentioned that none of these vectors has any effect on reporter gene
expression from control plasmid pTK-LUC (not shown). The cells were
incubated with or without T3. Cells harboring a
vector expressing ß-galactosidase cDNA (pRSVßgal) were included as
a control. The results are presented in Fig. 4b
. In the presence of
T3, as expected, a 3-fold inhibitory effect is
observed using p164PRL-LUC only when the thyroid hormone receptor is
coexpressed. In the absence of TR, transcription was stimulated
approximately 20-fold in the presence of c-jun and
c-fos regardless of whether T3 was
present. In contrast, stimulation by c-jun and
c-fos was nearly abolished when both
T3 and its receptor were present. Furthermore,
mutation of the consensus AP-1 site in p164(AP-1)mPRL-LUC completely
abolished both c-jun/c-fos activation and
T3 inhibition of the hPRL promoter in HeLa
cells.
These results strongly suggest that AP-1 is able to activate the hPRL promoter and that T3 exerts its inhibitory effect via an interaction between the hormone-bound receptor and the AP-1 complex.
AP-1 Binds to the hPRL Proximal Promoter
To confirm the involvement of AP-1 in hPRL expression, we
performed gel retardation assays. The -64/-35 fragment was used as a
probe in the presence of AP-1-enriched HeLa cell extracts. The results
are presented in Fig. 5. One retarded complex was
obtained (lane 2), which disappeared in the presence of a 100-fold
excess of an oligonucleotide containing the AP-1 consensus sequence
(lane 3), but not in the presence of the nonspecific DR4
oligonucleotide in a 100-fold excess (lane 4). In addition, the complex
disappeared in the presence of antibodies recognizing the DNA-binding
region of c-jun (lane 5). All of these data show that the
observed band is a specific AP-1 DNA complex.
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T3 Abolishes Estradiol (E2) Stimulation of
the hPRL Promoter
The transfection experiments using 5'-deletion mutants of the hPRL
promoter suggested the presence of a modulatory element in the distal
region (-1330/-740) that leads to a decrease in the overall
T3 inhibition. Preliminary studies identified a
complex element centered at -1200 that is able to bind TR and, in
addition, contains an estrogen response element (34). To further
analyze the role of this distal regulatory region, we examined the
effects of thyroid hormone and estrogen on a fragment of the hPRL
promoter containing both the proximal promoter and the distal
regulatory region (p2627PRLCAT; Fig. 6). The results
show, as expected, that T3 exerts an
approximately 3-fold inhibitory effect, and E2 has a 2-fold
stimulatory effect. In cells treated with both hormones, the recorded
CAT activity is surprisingly low, reaching only half the basal level.
In conclusion, the impact of the distal element on
T3 regulation appears to be weak, even when it is
stimulated by estrogens.
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DISCUSSION |
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Our finding that T3 inhibits the hPRL promoter is in keeping with the physiological data on the endogenous human gene. Hypothyroidism in human patients is frequently associated with increased serum PRL levels, whereas hyperthyroid patients present lower PRL levels than euthyroid control subjects (40). Although it is unclear whether this effect is due to direct regulation of the hPRL gene, recent studies on a human pituitary cell line suggest 2-fold inhibition of endogenous hPRL gene transcription by T3 (41).
The Proximal Promoter
The proximal hPRL promoter mediates T3
inhibition of a heterologous promoter (TK) in both pituitary
(GH3B6) and nonpituitary cells (HeLa, CV1, and
JEG-3; Fig. 2). On the other hand, we observe no binding of TR to the
proximal T3 response element in gel retardation
assays (Fig. 3
). This suggests that T3 acts
indirectly. Recently, T3 treatment was shown to
inhibit Pit-1 gene transcription through interference between the
thyroid hormone receptor and Pit-1 (42). As T3
inhibition of the hPRL promoter is observed in nonpituitary cells (Fig. 2c
), we can exclude an indirect action of T3
through inhibition of Pit-1 expression. Furthermore, coexpression of
Pit-1 and TR does not affect T3 inhibition in
HeLa cells (Fig. 2c
), so we can rule out a mechanism involving
interference between the TR and Pit-1 leading to inhibition of the hPRL
gene promoter, nor is the forskolin pathway involved in the response of
the hPRL promoter to T3 (our data not shown).
This contrasts with T3 inhibition of Pit-1 gene
expression, which involves interference of T3
with the cAMP induction pathway (42).
An AP-1-responsive element has been located in the hPRL proximal
promoter between coordinates -61 and -54 (see also Caccavelli, L., I.
Manfroid, J. A. Martial, and M. Muller, in preparation). It contains an
AP-1-binding sequence that differs by only one mismatch from the
consensus AP-1-binding site. We show that this sequence can
specifically bind AP-1 in gel retardation assays (Fig. 5) and that it
mediates 15-fold stimulation of reporter gene transcription in the
presence of coexpressed c-jun and c-fos (Fig. 4b
). This stimulation is almost completely abolished in the presence of
hormone-bound TR. We conclude that thyroid hormone exerts its
inhibitory effect via interaction of hormone-bound TR with AP-1.
Furthermore, mutation of the AP-1 site completely abolishes the
stimulatory effect of c-jun and c-fos and the
inhibitory effect of T3 (Fig. 4
). This confirms
that the two responses are interrelated.
The transcription-stimulating activity of AP-1 is increased by protein kinase C phosphorylation. Gellersen et al. (34) observed synergistic stimulation of the hPRL proximal promoter (250 bp) by TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment and Pit-1 expression in the SKUT-1B-20 human uterine cell line. Here we show strong stimulation of the hPRL promoter in HeLa cells cotransfected with a reporter construct and with c-jun and c-fos expression vectors, but this effect is completely Pit-1 independent. This discrepancy suggests that TPA stimulation in SKUT 1B-20 cells might involve another mechanism(s) in addition to the increased AP-1 activity.
Several groups have reported interaction of AP-1 with members of the nuclear receptor family, e.g. the glucocorticoid, retinoic acid, progestin, estrogen, and thyroid hormone receptors (15, 16, 17, 18, 19, 20, 21, 22). Different mechanisms have been observed for this cross-modulatory action according to the promoter context (22). Direct interaction between TR and AP-1 has been demonstrated in coimmunoprecipitation assays (20). Furthermore, in gel retardation experiments, binding of AP-1 to DNA was inhibited by TR when the latter was added before addition of the labeled binding sequence (19, 20). The hPRL proximal promoter contains one AP-1-responsive element and no TR-binding site. This suggests that TR blocks the action of AP-1 by hindering its binding to its response element. Whether the interaction between TR and AP-1 is direct or mediated by another factor(s) is unclear.
In striking contrast to the overall inhibition of hPRL transcription by T3, the combined positive and negative acting elements of the rPRL promoter mediate overall activation of this promoter by T3 in GH3 cells (39). The rPRL promoter responds differently to T3 according to the pituitary cell line examined (36, 37, 38, 39). Nevertheless, studies in rats have shown that treatment with the antiarhythmic drug amiodarone, a T3 antagonist, reduces PRL mRNA levels (43), suggesting that rPRL expression is up-regulated by T3. The rat sequence corresponding to the hPRL proximal AP-1 sequence differs by three nucleotides from the AP-1 consensus binding sequence, suggesting that rat and human PRL genes might be differently regulated by AP-1. Indeed, recently it was shown that expression of c-jun inhibits rat PRL expression in GH4 pituitary cells (44). This repression involves the FPII region centered at -125 in the rat promoter and is only seen in GH4 cells. The proposed mechanism is an indirect binding of c-jun to the promoter by recruitment of a pituitary-specific FPII-binding factor. Whether T3 would be able to block this repression, which would result in an overall activation by T3, is at present unknown. These observations contrast with our results using the hPRL promoter in GH3B6 cells. However, the same researchers describe a synergistic activation of the rPRL promoter by c-jun and Pit-1 expression in HeLa cells. In this case, the proposed mechanism is an interaction of c-jun with Pit-1 bound to the proximal binding site, but again without direct DNA binding of c-jun. Nothing is known about T3 modulation of this effect, which is suppressed in GH4 cells by the c-jun repression described above. Thus, it seems very likely that the mechanism of T3 inhibition of the hPRL promoter described here does not apply to the homologous rPRL promoter.
Complete hPRL Promoter
The combined action of the proximal, negative promoter and the
distal, weakly positive region results in inhibition by
T3 of the complete upstream region. In addition,
this region contains a consensus estrogen-responsive element (ERE)
located close to a TR-binding sequence (see also Van de Weerdt, C., F.
M. Pernasetti, L. Caccavelli, J. A. Martial, and M. Muller, in
preparation). The same hPRL ERE can mediate 2-fold stimulation of a
heterologous promoter in SKUT-1B-20 cells, a PRL-producing uterine
carcinoma cell line (34). The fact that T3 is
also able to block E2 activation of this construct (Fig. 6)
stresses the importance of the T3 regulation
mediated by the proximal AP-1 site.
Activation of rPRL gene transcription by E2 involves the stabilization of a chromatin loop permitting critical interactions between proteins of the distal enhancer and proximal promoter (45). Dexamethasone-bound GR has been found to bind to the distal ERE, competing in this with estrogen receptor and preventing formation of the loop (46). We have no information as to whether a particular chromatin structure characterizes the plasmid constructs used here, but hindering of loop formation by TR is certainly a possible mechanism of repression, especially as factors bound to the proximal promoter are known to be important in loop formation. If the AP-1 complex plays a role in the interactions between the distal and proximal regions, then T3 blocking of AP-1 binding to the DNA would inhibit loop formation, thus preventing E2 stimulation. Alternatively, T3 might down-regulate the expression of other proteins necessary for the response to estrogen mediated by the complete hPRL promoter.
In conclusion, we demonstrate that hPRL gene transcription is inhibited by thyroid hormone. We show that the inhibitory effect of T3 involves interference with the AP-1 transactivation mediated by an AP-1-binding site located in the proximal hPRL promoter. A weak positive TRE, located in the distal region close to an ERE, modulates this inhibition. However, T3 treatment reduces the E2 stimulatory effect at a promoter region containing both proximal and distal response elements. Thus, our work illustrates the complexity of mechanisms involved in gene transcriptional regulation. Cross-talk between nuclear receptors and other transcription factors provides an intricate set of distinct regulatory mechanisms permitting precise control of a specific gene expression by intra- and extracellular factors.
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MATERIALS AND METHODS |
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Oligonucleotides
The double or single stranded oligodeoxyribonucleotides used for
plasmid construction or gel mobility assays were obtained from
Eurogentec (Seraing, Belgium). The AP-1 oligonucleotide contains the
AP-1 site of the human collagenase promoter (56). The numbers represent
positions in the PRL upstream sequence, where relevant.
For each oligonucleotide the sequence of one strand is presented: -250PRL, 5'-CCCAAGCTTAGATCTCACCTTTCAAC-3'; -35PRL, 5'-GCTCTAGATATCTTCATGAATATAATG-3'; DR4, 5'-TCGAAGCTTCAGGTCACAGGAGGTCAA-GCT-3'; AP-1 m, 5'-CTTCATGAATATAATCGAGCAGGCATTCGTTTCCC-3'; and Sp1, 5'-AGTTCCGCCCATTCTCC-GCCCCA-3'.
Cell Cultures and Transient Transfection
GH3B6 and HeLa cells were grown in
monolayers in DMEM supplemented with 10% FCS plus E2 (5 x
10-10 M). Twenty-four hours before
transfection, the cells were grown in phenol red-free DMEM supplemented
with 2% AG1X8 resin-charcoal-stripped FCS (52).
T3, E2, and forskolin were
obtained from Sigma (Deisenhofen, Germany).
Electroporation
GH3B6 cells were harvested with trypsin-EDTA
and resuspended in phenol red-free DMEM with 2% depleted FCS (FCSt;
final concentration, 2 x 106 cells/ml). Thirty
micrograms of each plasmid were mixed with 1.6 x 106
cells. The cells were then exposed to a single pulse of 250 V/4 mm and
1500 µF capacitance with an Easyject electroporator (EquiBio,
Seraing, Belgium). Transfected cells were immediately transferred to
phenol red-free DMEM with 2% FCSt and incubated for 48 h.
Treatment of the electroporated cells is detailed in the figure
legends.
Calcium Phosphate Precipitation
HeLa cells were transfected by the CaPO4 method, as
previously described (53). The day before transfection, 5 x
105 cells were plated in petri dishes (9 cm in diameter) in
phenol red-free DMEM with 2% FCSt. Two to 10 µg reporter plasmid
were mixed with 5 µg of each expression vector specified; the total
amount of expression vector was adjusted to 20 µg by the addition of
the appropriate amount of pRSVßgal. After a 12-h incubation with
CaPO4 precipitate, the cells were washed with PBS and
incubated in phenol red-free DMEM with 2% FCSt for 48 h. Hormone
treatments are detailed in the figure legends.
CAT Assay
After a 48-h incubation, the cells were harvested by scraping and
resuspended in 100 µl 250 mM Tris-HCl (pH 7.6). Cell
disruption and the CAT assay were performed as described previously
(32).
Luciferase Assay
After a 48-h incubation, the cells were harvested by scraping and
directly resuspended in lysis buffer. The assays were performed as
described previously (54).
Protein concentrations in extracts were determined by the Bradford assay. Fifty micrograms of total extract were used in both the CAT and LUC assays.
Mobility Shift Assays
Oligonucleotides and PCR fragments were [32P]ATP
labeled using T4 polynucleotide kinase. Labeled probes were purified by
elution from polyacrylamide gels. Proteins were preincubated in a
buffer containing 20 mM HEPES (pH 7.8), 4 mM
MgCl2, 2 mM dithiothreitol (DTT), 10%
glycerol, 0.2 mg/ml BSA, and 80 mM KCl with 1 µg
poly(dI-dC) for 30 min at 4 C. When necessary, antibodies were
preincubated with proteins for 30 min. After preincubation, 10,000 cpm
probe were added, and incubation proceeded for 30 min at 4 C. The
resulting protein-DNA complexes were resolved by electrophoresis on a
prerun 5% polyacrylamide gel with 0.5 x TBE as the running
buffer for 2 h at 4 C. The gel was dried and autoradiographed
overnight. Polyclonal rabbit anti-Jun antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). The antigen sequence used to
produce the antibodies corresponds to residues 247263 within the
C-terminal DNA-binding domain of the mouse c-Jun protein. Polyclonal
rabbit antithyroid hormone receptor 1 antibody was purchased from
Affinity Bioreagents (Neshanic Station, NJ). The antigen sequence used
to produce the antibodies corresponds to residues 403410 of the
extreme C-terminal region of the human TR
, which is identical to the
rat sequence. Recombinant RXR was provided by Dr. Baes, University of
Leuven (Leuven, Belgium).
Bacterial Expression and Purification of hTR1
The bacterial strain BL21(DE3)pLYS S (55) was transformed with the
vector pET-hTR1 (provided by L. J. De Groot). A freshly transformed
clone was used to inoculate 500 ml Luria Bertoni medium. The culture
was grown at 37 C to an OD of 0.4,
isopropyl-ß-D-thiogalactoside (IPTG) was added to a
0.5-mM final concentration, and the culture was gently
rocked for 5 h at 20 C. The bacteria were harvested by
centrifugation and resuspended in 20 ml lysis buffer (20 mM
Tris, pH 8.0; 100 mM NaCl; 0.5 mM
MgCl2; 1 mM phenylmethylsulfonylfluoride; and
1% aprotinin). The suspension was sonicated on ice to obtain a clear
lysate; 2 ml of a mixture of 0.1 M DTT, 0.5 M
EDTA, and 5% Nonidet P-40 were added; and bacterial debris were
removed by centrifugation at 60,000 x g. Soluble proteins
were precipitated from the supernatant by the addition of 0.33 g/ml
ammonium sulfate and recovered by centrifugation at 60,000 xg. The pellet was resuspended in 1 ml HS buffer (25 mM
HEPES-KOH, pH 7.6; 5 mM MgCl2; 1 mM
EGTA; 1 mM DTT; and 10% glycerol), and the solution was
dialyzed against HS buffer. The fraction was loaded on a
heparin-Sepharose column, and proteins were eluted with a linear KCl
gradient. Human TR
1 was detected in the fractions by means of a gel
retardation assay using a DR-4 probe and by SDS-PAGE followed by
Coomassie staining or Western blotting. Eluates at 0.40.7
M KCl contained greater than 95% pure, soluble,
bacterially expressed hTR
1. We used 4 µg purified TR extract in
each gel retardation assay unless otherwise indicated in the figure
legends.
HeLa Cell Extracts Enriched with AP-1 Complex
HeLa cells were transfected with 25 µg pRSVc-jun and
pRSVc-fos according to the diethylaminoethyl-dextran
transfection protocol (56). The cells were harvested 48 h after
transfection. Total protein extracts were prepared by resuspending the
cells in 20 mM HEPES, pH 7.8; 400 mM KCl; 20%
glycerol; and 2 mM DTT. They were disrupted by three
freeze-thaw cycles, followed by centrifugation at 12,000 rpm for 30
min. Total enriched extract was added in each gel retardation assay in
an amount corresponding to 4.5 µg protein, as determined by the
Bradford assay.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from the Services Federaux des Affaires Scientifiques, Techniques et Culturelles (PAI P3042 and PAI P3044); Fonds National de la Recherche Scientifique (3.4537.93 and 9.4569.95); and Actions de Recherche Concertes (95/00193).
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FOOTNOTES |
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1 Present address: Department of Reproductive Medecine, 9500 Gilman
Drive, University of California-La Jolla, San Diego, California
92093-0674.
2 Fellow of the National Council for Scientific and Technological
Development, Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico, Brazil.
Received for publication May 20, 1996. Revision received December 30, 1996. Revision received March 14, 1997. Accepted for publication March 14, 1997.
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REFERENCES |
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