From the George Whipple Lab for Cancer Research,
Departments of Pathology, Urology, Radiation Oncology, and the Cancer
Center, University of Rochester Medical Center, Rochester, New York
14642 and the ¶ Department of Molecular Microbiology and
Immunology, University of Southern California, Los Angeles, California
90033
Received for publication, June 14, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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The TR4 orphan receptor is a member of the
nuclear receptor superfamily that modulates gene expression via binding
to the AGGTCA direct repeat hormone response element. Here we report a
functional study of TR4 interaction with the core promoter of the
hepatitis B virus (HBV). The electrophoretic mobility shift assay shows
that TR4 can bind to the direct repeat 1 sequence element
(AGGTTAAAGGTCT, nucleotide coordinates
1757-1769, TR4RE-HBV) on the HBV core promoter. TR4 also can enhance
the activity of a synthetic luciferase reporter linked with four copies
of TR4RE-HBV in either liver HepG2 or non-liver H1299 cells in a
dose-dependent manner. Surprisingly, TR4 represses the
activity of a luciferase reporter containing the entire HBV genome
sequences. Moreover, mutation of this TR4RE-HBV site in the HBV core
promoter diminishes the TR4 suppression effect. This TR4-induced
suppression of HBV core promoter activity is further confirmed by
primer extension analysis of the HBV core RNAs, showing that TR4
represses both pre-core and core mRNAs. Further dissection
of this repressive mechanism indicates that TR4 may suppress the HBV
core promoter activity via repressing HNF4 Hepatitis B virus (HBV)1 is a small virus with a
3.2-kb partially double-stranded DNA
genome, containing four open reading frames: the surface antigen, the
core antigen, the polymerase, and the X protein (1, 2). HBV is a major
pathogen causing acute and chronic liver diseases, such as cirrhosis
and hepatocellular carcinoma (3, 4). HBV transcripts are regulated by
the transcriptional control of four different promoters and two
enhancer elements (5). HBV is replicated from a RNA intermediate as a
template for reverse transcription (1). The core promoter controls the expression of the 3.5-kb core mRNA (C mRNA) as the template for replication, and the expression of the pre-core mRNA (pre-C mRNA) for the translation of the HBeAg precursor (6). Recently, multiple binding sites for liver-specific or ubiquitous transcription factors and several hormone response elements (HRE) have been identified in the
core promoter region, including Sp1 (7, 8), TATA-binding protein (9),
HNF3 (10), HNF4 The testicular orphan receptor 4 (TR4) (NR2C2) is an orphan member of
the nuclear receptor superfamily (15-17). This family includes classic
steroid hormone receptors, the thyroid hormone receptor, the retinoid
receptors, and many nuclear orphan receptors whose physiological
ligands have not been identified (18). Members of this superfamily
typically contain two major conserved domains, the DNA-binding domain
(DBD) and the ligand-binding domain (LBD) (18). Most nuclear receptors
bind to their cognate ligands, translocate into the nucleus, and bind
to the specific HREs to turn on their downstream target genes. TR4 was
isolated from human prostate and testis cDNA libraries and is
considered to be an orphan receptor, because its ligand has not been
found (15). Our previous data showed that TR4 recognizes different
AGGTCA direct repeat (DR) sequences either inducing or suppressing its target genes. For example, TR4 induces reporters containing the DR1
site of human ciliary neurotrophic factor The distribution of TR4 expression is ubiquitous in many tissues,
including the central nervous system, adrenal gland, spleen, thyroid
gland, and liver (16, 19, 23). Also, there are many other nuclear
receptors expressed in the liver, such as HNF4 In this report, we have analyzed the DR1 site in the HBV core promoter.
We tested whether the TR4 plays a role in regulating the activity of
the core promoter through binding to the HRE-DR1 site. We showed that
TR4 binds to the DR1 site in the HBV core promoter and strongly
activates a reporter containing synthetic DR1 response elements.
Moreover, we demonstrated that TR4 could suppress both pre-C and C RNA
expression, which was also confirmed by the luciferase assay using a
reporter containing the entire core promoter. Our data further suggest
that TR4 may suppress HBV core promoter activity via the interruption
of HNF4 Plasmids--
The plasmids CpFL(4)LUC, CpLUC, XpHNF4LUC, and
CpM2LUC were gifts from Dr. A. McLachlan (The Scripps Research
Institute) and were described previously (13). The plasmid
pCDNAI-HNF4 Electrophoretic Mobility Shift Assay--
The EMSA reaction was
performed as described previously (20). Briefly, TR4 and HNF4 Cell Culture, Transfections, and Luciferase Assays--
HepG2
cells, Huh7 (human differentiated hepatoma cells), H1299 cells, or
HepG2.1 cells (human de-differentiated hepatoma cells) were maintained
in Dulbecco's modified Eagle's medium or RPMI supplemented with 10%
fetal bovine serum, 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin (31). The cells were plated at
105/well in 12-well dishes (Falcon) and transfected with
1.2 µg of DNA/well using SuperFect according to the manufacturer's
protocol (Qiagen). The internal control plasmid pRL-TK (Promega) was
co-transfected in all transfection experiments. After 48 h of
transfection, the cells were harvested, and luciferase assays were
performed using the Dual-Luciferase kit (Promega). The HBV transfection
experiment was carried out as described previously (8).
Primer Extension Assay--
The primer to map the C gene
transcripts was 5'-GGTGAGCAATGCTCAGGAGACTCTAAGG-3', which corresponds
to coordinates 2024-2051 of the HBV genomic sequence. The reaction was
performed as previously described (32). Briefly, The HBV DNA
pWTD containing the wild type HBV genome (adw2) was
co-transfected with the nuclear receptors into Huh7 hepatoma cells
using the calcium phosphate precipitation method. The pXGH vector
served as an internal control for the normalization of transfection
efficiency. After 48 h of transfection, the RNA was isolated for
the primer extension assay (32).
GST Pull-down Assay--
The GST-TR4 fusion protein and GST
protein were purified by glutathione-Sepharose beads according to the
manufacturer's instructions (Amersham Biosciences). HNF4 Co-immunoprecipitation of TR4 and HNF4 TR4 Protein Binds Specifically to the TR4RE-HBV Consensus Site of
the HBV Core Promoter--
Previous studies showed that the activity
of the HBV core promoter could be differentially regulated by RXR
To further examine whether endogenous TR4 protein can interact with the
TR4RE-HBV, nuclear extracts isolated from human HepG2 cells were used
in the same EMSA. As shown in Fig. 2, no
supershift complex was seen when the nuclear extracts were incubated
with the double-mutant M1 probe in the presence of an anti-TR4 antibody (Fig. 2, lanes 1 and 2). The wild type TR4RE-HBV
probe clearly binds with TR4 as indicated by the
TR4·TR4RE-HBV·antibody supershift complex (lanes 3 and
4). This result shows that TR4 protein is present in liver
cells and interacts with TR4RE-HBV, suggesting that this interaction
may play a role in regulating HBV core promoter activity.
TR4 Enhances the Activity of a Luciferase Reporter Linked to
TR4RE-HBV in Liver HepG2 and Non-liver H1299 Cells--
Our previous
data showed that the DR1 consensus sequence (RXRE) from the
promoter of cellular retinol-binding protein II could be recognized and
suppressed by TR4 (21). We were interested in determining whether the
binding of TR4 to the TR4RE-HBV could also allow TR4 to regulate HBV
core promoter activity. A reporter construct containing four synthetic
copies of the TR4RE-HBV, CpFL(4)Luc, including the HNF4 TR4 Directly Suppresses HBV Core Promoter Activity through the
TR4RE-HBV Site--
The EMSA and reporter assay with CpFL(4)Luc
suggest that TR4 may have a significant role in the transcriptional
regulation of the HBV core promoter. However, the functional role of
TR4 in a synthetic promoter context does not necessarily reveal TR4 action in the natural HBV core promoter context. We therefore examined
the effects of TR4 on a reporter construct containing the whole context
of the HBV genome sequence (CpLuc) (13). Unexpectedly, TR4 suppressed
CpLuc reporter activity in HepG2 cells (Fig.
4A). In contrast,
co-transfection of another nuclear receptor, HNF4
Because the responsiveness of the HBV core promoter to TR4 was
abrogated by this mutation, it is possible that the TR4-mediated suppression of the HBV core promoter is mainly dependent upon the
binding between TR4 and the TR4RE-HBV. Because earlier reports and our
current data show that both TR4 and HNF4
Using EMSA with in vitro translated TR4 and HNF4 Interaction between HNF4
Next, we hypothesized that TR4 may directly regulate HNF4
In summary, the results presented above demonstrate that TR4 is capable
of regulating HNF4 Suppression of HBV Pre-core and Core mRNA Expression by
TR4--
To further confirm the suppression of HBV core promoter
activity by TR4, we applied a primer extension assay to see if TR4 can
also influence mRNA expression of two HBV transcripts from the HBV
core promoter. The first transcript is pre-C mRNA, which is the
precursor of HBeAg; the other transcript is C mRNA, serving as the
template for reverse transcription (6, 36). HBV RNA extracted from
Huh-7 after the co-transfection of TR4 and HBV vectors was mapped with
radiolabeled HBV primer at position 2024-2051 of the HBV genome (8).
As seen in Fig. 7, the expression of both
pre-C and C mRNAs was decreased in the presence of TR4. This result
therefore confirms the reporter assays showing that TR4 can suppress
HBV core promoter transcription via binding to the TR4RE-HBV site on
the HBV core promoter.
Earlier studies have identified two consensus DR1 binding sites
for nuclear receptors in the HBV promoter. One such site
(TGAACCTTTACCC, nucleotide coordinates
1138-1150), is located in the core domain of enhancer I and is
recognized by HNF4 TR4 is highly homologous with another orphan receptor, TR2 (15).
Earlier studies indicated that TR2 and TR4 could modulate the
expression of a set of genes through binding to the same HREs (19, 38).
Interestingly, Yu et al. (14) reported that TR2 could only
repress pre-C RNA, but not C RNA expression. Our current data show that
TR4 can repress both pre-C RNA and C RNA expression. Moreover, compared
with the marginal suppression of TR2 on CpLuc reporter activity, TR4
has a much stronger suppressive effect on CpLuc reporter activity (data
not shown), suggesting that TR4 may play a significant role in this
regulation. Also, our data suggest that simple competition for binding
to the DR1 site by these two nuclear receptors may not fully account
for their distinct regulation of HBV core gene expression, because both
nuclear receptors regulate target genes through binding to the DR sites
(19, 38). The detailed mechanisms underlying the distinct regulation of HBV gene expression by these two closely related orphan receptors remain unclear. The interaction of TR4 and HNF4 The contrasting effects of TR4 on the synthetic TR4RE-HBV
versus the entire HBV promoter (Fig. 3 versus 4)
suggest that promoter context, especially sequences other than the
TR4RE-HBV, may also play important roles for TR4 to modulate HBV core
promoter activity. It is possible that sequences flanking the TR4RE-HBV
may recruit one or more repressors that can cooperate with TR4 to form
a protein complex to repress HBV core promoter. In addition, we have
examined the effects of TR4 on HBV core promoter activity in non-liver cells and found that TR4 was able to induce the luciferase activity of
the Cpluc reporter in H1299 cells (data not shown). This interesting finding implies that some cellular factors in liver cells may modulate
the transcriptional activity of TR4 in the regulation of the HBV core
promoter. However, the detailed mechanisms of this distinct regulation
by TR4 remain unclear.
TR4 may modulate its target gene expression via two distinct
mechanisms. First, TR4 can suppress target gene expression via competition with other nuclear receptors, such as RXR On the other hand, in HBV-infected liver cells, the repressive function
of TR4 may be unfavorable to the HBV replication because the core
promoter controls the transcription of the template C RNA for viral DNA
synthesis. We speculate that the functional association of TR4 and
HNF4 As summarized in Fig. 8, TR4 can directly
bind to the TR4RE-HBV and therefore prevent other nuclear receptors
from activating the HBV core promoter. Alternatively, TR4 can interact
with nuclear receptors, such as HNF4-mediated transactivation
by protein-protein interactions without inhibition of HNF4
DNA
binding. Furthermore, our results indicate that the N- and C-terminal
regions of TR4 protein are required for TR4-HNF4
interaction. It is
possible that TR4-HNF4
interaction may block the HNF4
function
that results in the suppression of HBV gene expression. Together, these
results demonstrate that TR4 can serve as a negative modulator in the transcriptional regulation of HBV core gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(11), C/EBP (12), and other members of the nuclear
receptor superfamily, such as the retinoid X receptor (RXR
),
peroxisome proliferator-activated receptor (PPAR
), COUP-TF1, and
ARP1 (13). For example, HNF4
and RXR
/PPAR
can stimulate the
expression of the 3.5-kb core RNA, COUP-TF1 suppresses the expression
of both the pre-C RNA and C RNA, and TR2 preferentially represses the
expression of the pre-C RNA (14). These findings suggest that these
nuclear receptors may play important roles in viral transcription. The
detailed mechanisms of how the core promoter is differentially
regulated by these nuclear receptors remain unclear.
receptor promoter and the
DR4 site of the
-myosin heavy chain promoter (19, 20). In contrast,
TR4 suppresses RXR
/retinoic acid receptor (DR1), vitamin D3 receptor
(DR3), and RXR
/PPAR
(DR1)-mediated transactivation as shown by
the reporter assay (21-23). These results suggest that TR4 may act as
a negative regulator of its target genes containing hormone response
elements (HREs) by competition with other nuclear receptors for binding
to the HREs (21, 23). In addition, our previous data showed that TR4
could also suppress androgen receptor-mediated transactivation via
protein-protein interaction (22).
(24) and
RXR
/PPAR
(25, 26). These findings suggest that these nuclear
receptors in the liver can coordinately regulate gene expression by
interacting with HRE sequences in the promoters of their target genes.
Our data show that TR4 can interfere with a number of other nuclear
receptors by competing for binding to the same HREs (21, 22).
-induced HBV core promoter by protein-protein interaction
with HNF4
. These data demonstrate that TR4 can function as a
repressor in liver cells to suppress HBV gene expression and replication.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was a gift from Dr. M. Hadzopoulou-Cladaras (Boston
University, Boston, MA). pG4LUC was a gift from Dr. K. L. Guan
(University of Michigan Medical School, Ann Arbor, MI). The pCMX-TR4,
pET14b-TR4, pSG5-VDR, and pCMV-TR2 plasmids were constructed as
described previously (27, 28). pWTDHBV contains the head-to-tail dimer
of the wild type HBV genome DNA (adw2) (8). The GST-TR4,
TR4-N-DBD, and TR4-LBD fusion constructs were described in our previous
study (29). The GAL4-HNF4
expression plasmid for one-hybrid assay was made by inserting the PCR-generated
BamHI/XbaI fragment (amino acids 1-465) of
HNF4
from pCDNAI-HNF4
into pM containing the GAL4
DBD-(1-147) (Invitrogen).
proteins for EMSA were transcribed and translated in TNT
reticulocyte lysates (Promega), according to the manufacturer's
instructions. The oligonucleotide probes for EMSA were end-labeled with
[
-32P]ATP by T4 polynucleotide kinase (New England
BioLabs). HepG2 cell nuclear extracts were prepared according to the
procedure of Andrew and Faller (30). The EMSA reactions were performed with 10 µg of nuclear extracts or 2 µl of TNT lysates
in an EMSA buffer containing 10 mM Hepes (pH 7.9), 6%
(v/v) glycerol, 2% (v/v) Ficoll, 100 mM KCl, 0.5 mM EDTA, 2.5 mM MgCl2, and 1 mM dithiothreitol. The reaction mixtures of the proteins
and DNA were incubated for 15 min at room temperature. For the antibody supershift assay, the mixtures were incubated for another 15 min in the
presence or absence of mouse anti-TR4 monoclonal antibody (#15). The
protein·DNA complexes were analyzed on a 5% native polyacrylamide gel.
was
synthesized using the in vitro TNT translation
system (Promega) in the presence of [35S]methionine, and
5 µl of HNF4
protein was incubated with GST-TR4, TR4-N-DBD,
TR4-LBD, or GST beads for the GST pull-down assay as described
previously (22).
Proteins--
COS1
cells were cultured in Dulbecco's modified Eagle's medium and
co-transfected with 10 µg of pCMX-TR4 and either 10 µg of
pCDNAI or pCDNAI-HNF4
. 48 h after the transfection, the
cells were lysed in immunoprecipitation buffer (10 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA,
0.5% sodium deoxycholate, and 0.5% Nonidet P-40) containing 1 µg/ml
protease inhibitors (Roche Molecular Biochemicals) on ice for 30 min.
The cellular lysates were centrifuged at 12,000 × g
for 10 min. The protein concentration was measured using the
Bradford reagent (Bio-Rad), 500 µg of protein was precleared using
protein A/G-Sepharose beads (Santa Cruz Biotechnology), and then the
mixture was immunoprecipitated with either an anti-TR4 monoclonal
antibody or normal mouse IgG (1:100) at 4 °C overnight. Protein A/G
beads were added, and the protein lysates were rotated at 4 °C for
another hour. The immunocomplexes bound by beads were pelleted and
washed with immunoprecipitation buffer three times and then subjected
to SDS-PAGE for Western blot analysis using a goat anti-HNF4
polyclonal antibody (Santa Cruz Biotechnology). The results were
visualized by enhanced chemiluminescence (ECL). The same blot was
stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS at 65 °C
for 30 min and then blotted with an anti-TR4 monoclonal antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or
PPAR
via recognizing consensus binding sites on the HBV core
promoter (13, 14). Our previous data also showed that the TR4 has a
better affinity than RXR
to recognize the DR1-HRE in the promoter of the cellular retinol-binding protein II gene (21). To examine whether
TR4 can bind to the potential DR1-HRE (named TR4RE-HBV, nucleotide
coordinates 1751-1778 of the HBV ayw DNA sequence) on the
HBV core promoter (13), the electrophoretic mobility shift assay (EMSA)
was performed with in vitro translated TR4 protein, using
radiolabeled TR4RE-HBV oligonucleotides as probes. As shown in Fig.
1, no specific supershift complex was
seen with an anti-TR4 monoclonal antibody when using mock lysates (Fig. 1, lanes 1 and 2). In contrast, the TR4-CpFL
supershift complex was observed in the presence of an anti-TR4 antibody
using in vitro translated TR4 lysates (lanes 3 and 4). Moreover, when we replaced wild type TR4RE-HBV with
various mutants, we found that TR4 could only bind specifically to the
wild type TR4RE-HBV, but not to other mutated TR4RE-HBVs, except for
the M3 mutant, which has marginal binding (lanes 5-10). The
M1 double mutant with nucleotides changed from A to T (1765) and G to A
(1767) represents the HBV mutant frequently found in the patients with
chronic hepatitis (33). TR4 was not able to bind to the M1 and M2
mutants (lanes 5 and 6), indicating that the
second mutated nucleotide of the DR1-AGGTCA motif is essential for TR4
binding to the TR4RE-HBV. In addition, a TR4-specific antibody can
supershift the TR4·TR4RE-HBV complex (lane 4), and this
binding can be reduced in the presence of 100-fold unlabeled TR4RE-HBV
probe but not the probe with an androgen response element probes or
TR4RE-HBV mutant probes (lanes 11-15), suggesting that the
binding between TR4 and TR4RE-HBV is specific.
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Fig. 1.
TR4 binds to the CpFL element of the HBV core
promoter in vitro. TR4 proteins were in
vitro synthesized in TNT reticulocyte lysates
(Promega) and then incubated with CpFL or CpFL mutant probes for 30 min
in the presence or absence of an anti-TR4 monoclonal antibody or
various 100-fold unlabeled probes as indicated. The reaction mixtures
were resolved on a 5% acrylamide native gel, and results were
visualized by autoradiography. ARE denotes the androgen
response element.
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Fig. 2.
The nuclear orphan receptor TR4 is an
endogenous DR-1 binding protein in HepG2 cells. The CpFL probe
containing the DR-1 site (nucleotide coordinates 1751-1780) or the M1
mutant probe were incubated with 10 µg of protein of nuclear extracts
isolated from HepG2 cells. The EMSA reaction was carried out at room
temperature for 30 min in the presence or absence of anti-TR4 or
anti-HNF4 antibody. The reaction complexes were analyzed on 5%
acrylamide with 0.25× TBE gels, and the results were visualized by
autoradiography.
consensus
binding site, was used to determine the role of TR4 (13). CpFL(4)
plasmids were co-transfected with either the parental vector or a TR4
expression vector in liver HepG2, HepG2.1, and non-liver H1299 cells.
TR4 can transactivate CpFL(4)Luc activity in a
dose-dependent fashion in HepG2, HepG2.1, and H1299 cells
(Fig. 3, A, B, and
D). To confirm that the increase in reporter activity is not
due to nonspecific activation by TR4, we also used the TR4 deletion
mutants and VDR in this reporter assay. As shown in Fig. 3
(A-C) the activity of the reporter was only induced in the
presence of full-length functional TR4, but not in VDR or the
presence of TR4 mutants lacking the C terminus or N terminus,
suggesting that the CpFL (TR4RE-HBV) elements of the core HBV promoter
may be sufficient for TR4 to regulate HBV core promoter activity.
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Fig. 3.
Effects of TR4 on a reporter containing the
CpFL(DR1) element of the HBV core promoter. A, HepG2
cells were co-transfected with 500 ng of CpFL(4)Luc reporter and
increasing amounts of TR4 or VDR (100, 200, and 400 ng). The cells with
VDR transfection were treated with 100 nM vitamin D for
24 h, then harvested for the luciferase assay. The luciferase
assay was performed as described under "Experimental Procedures."
B, HepG2.1 cells were transfected with CpFL(4)Luc and TR4 or
VDR, treated, and harvested as in A. C and
D, HepG2 cells and H1299 cells were co-transfected with
CpFL(4)-Luc and the full-length TR4, N/C-terminal deletion of TR4
(TR4/delN and TR4/delC). The reporter assay was carried out as
described in A. Data represent the means ± S.E. of
three independent experiments.
, induced CpLuc
reporter activity, consistent with an earlier report (13). To confirm
that the suppression was mediated by TR4 through binding to the
TR4RE-HBV of the HBV core promoter, we also used CpM2Luc containing the
mutated TR4RE-HBV to analyze TR4 suppression (13). As shown in Fig.
4B, TR4 suppression of the reporter activity was abolished
by mutation of the TR4RE-HBV site. In contrast, HNF4
was able to
induce mutant CpM2Luc reporter activity, possibly due to the presence
of another functionally intact HNF4
-binding site (lane 4 versus 6) (13, 34).
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Fig. 4.
TR4 represses HBV core promoter
activity. A, HepG2 cells were co-transfected with 750 ng of CpLuc and 250 ng of TR4 or HNF4 vectors or the combination of
these two nuclear receptors. The luciferase activity was measured
48 h after the transfection. B, the reporters CpLuc
(750 ng) or CpM2Luc were co-transfected with TR4 (250 ng) or HNF4
(250 ng) as indicated, then harvested for the luciferase activity
48 h after the transfection. Data represent the mean ± S.E.
of three independent experiments. PSp, Sp,
Xp, and Cp represent the promoters of the genes
encoding the HBV surface antigen, X protein, and core/e antigens,
respectively.
can bind to the TR4RE-HBV
site, which is overlapped with the HNF4
consensus binding site (13),
we postulated that TR4 may exert its repressive effects on the core
promoter through competition with other nuclear receptors such as
HNF4
for binding to TR4RE-HBV. We therefore examined their potential
mutual influence on the HBV core promoter. When we co-transfected equal
amounts of HNF4
and TR4 with the CpLuc reporter in HepG2 hepatoma
cells, TR4 repressed HNF4
-induced HBV core promoter activity (Fig.
4A, column 3 versus 4),
suggesting that HNF4
and TR4 may compete for the same consensus
binding site on the HBV core promoter. Similar suppression of
HNF4
-mediated transactivation also occurred when we used another
reporter containing the HNF4
binding site found in the HBV enhancer
I/X promoter overlapped with enhancer I (XpHNF4
Luc, HBV coordinates
1133-1159, data not shown) (13).
in the
presence of [32P]TR4RE-HBV, we also found that the
binding between [32P]TR4RE-HBV and TR4 protein was
suppressed by the addition of HNF4
(Fig.
5). Fig. 5 demonstrates that TR4 and
HNF4
can both bind to the TR4RE-HBV site, and the DNA binding
activity of TR4 was markedly reduced in the presence of HNF4
. In
contrast, we did not observe any change in HNF4
binding when we
added TR4, implying that the simple competition may not fully account
for TR4-mediated repression of HNF4
function. This raises the
possibility that TR4 may directly block HNF4
transactivation through
protein-protein interactions with HNF4
instead of competing for DNA
binding sites. Together, Figs. 4 and 5 suggest that TR4 can bind to the
same TR4RE-HBV site as well as interact with HNF4
to influence HBV core promoter expression.
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Fig. 5.
HNF4 suppresses TR4
binding to the CpFL element of the HBV core promoter in
vitro. TR4 and HNF4
proteins were in
vitro synthesized in TNT reticulocyte lysates
(Promega). 1 µl of TR4 protein was then incubated with CpFL probes
for 15 min in EMSA reaction buffer (10 mM Hepes, pH 7.9/6%
(v/v) glycerol, 2% (v/v) Ficoll, 100 mM KCl, 0.5 mM EDTA, 2.5 mM MgCl2, and 1 mM dithiothreitol) in the presence or absence of the
HNF4
protein. The reaction mixtures were resolved on a 5%
acrylamide native gel, and results were visualized by autoradiography.
The double asterisks indicate that the repression of TR4 DNA
by HNF4
.
and TR4--
The data shown above
suggest that TR4 may regulate the transcriptional activity of HNF4
by a mechanism that is independent from the competition for DNA
binding. To prove this hypothesis, we constructed a plasmid encoding
GAL4-HNF4
fusion protein and examined the effect of TR4 expression
on HNF4
-mediated transactivation in the GAL4 one-hybrid system (35).
In this assay, the transcriptional activity of GAL4-HNF4
is
independent of the binding of HNF4
and TR4 to their cognate binding
site, DR1-HRE. If TR4 can directly regulate HNF4
transcriptional
activity, we should see the activity of GAL4-HNF4
would be
significantly changed in the presence of TR4 expression. HepG2 cells
were co-transfected with GAL4-HN4
and the pG4LUC reporter in
the presence or absence of the expression of the TR4 N- or C-terminal
deletion of TR4 mutants. As the results show in Fig.
6A, the reporter activity with
GAL-HNF4
alone was increased about 3-fold, which was similarly shown
by the previous study (35). Interestingly, the expression of TR4
significantly increased the GAL4-HNF4
-mediated transcriptional
activation (Fig. 6A), indicating that TR4 can directly
regulate the transcriptional activity of HNF4
, which is independent
of the event of the DNA binding and possibly mediated by
protein-protein interactions. As expected, the PG4LUC reporter activity
with the GAL4 and TR4 was not increased at any significant level (Fig.
6A, columns 1 versus 6),
suggesting TR4 influences HNF4
transcriptional activity in a
specific manner. Nevertheless, it is not clear how this regulation involving the interaction between TR4 and HNF4
would result in either activation or suppression of HNF4
function. In contrast, the
N- or C-terminal deletion of TR4 mutants did not show any effects on
the GAL4-HNF4
-mediated transactivation, suggesting that these two
regions of TR4 protein may be required for TR4 action in this
regulation.
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Fig. 6.
Interaction between TR4 and
HNF4 . A, diagrammatic
representation of the GAL4 hybrid assay. This assay was used to test
the effect of TR4 on HNF4
-mediated transcriptional activity. HepG2
cells were co-transfected PG4LUC, which contains four tandem repeats of
the GAL4-binding sites, with GAL4-HNF4
, TR4, or TR4 deletion
mutants. B, in vitro synthesized
[35S]methionine-labeled HNF4
protein was incubated
with GST, GST-TR4, GST-TR4-N-DBD, and GST-TR4-LBD fusion protein bound
to glutathione-Sepharose beads. The beads were extensively washed after
the reaction and applied to 12% SDS-PAGE followed by
autoradiography. C, COS1 cells were transfected pCMX-TR4
with either parental vector or pCDNAI-HNF4
. After the
transfection, the cells were cultured for 48 h, lysed for the
immunoprecipitation assay, and the HNF4
signal was detected by
Western blotting with a goat anti-HNF4
antibody. The results were
visualized by enhanced chemiluminescence (ECL). Then, the same blot was
stripped and blotted with an anti-TR4 monoclonal antibody.
D, HepG2 cells were transfected CpLUC with HNF4
, TR4, or
TR4 deletion mutants as described in Fig. 4A.
transcriptional activity through interacting with HNF4
. To test this
hypothesis, we applied the GST pull-down assay to determine the
potential interaction between HNF4
and TR4. As shown in Fig. 6B, HNF4
protein was pulled down and detected by SDS-PAGE
from the mixture of 35S-labeled in vitro
translated HNF4
protein incubated with GST-TR4, GST-TR4-N-DBD, or
GST-TR4-LBD fusion protein immobilized on glutathione-linked Sepharose
beads. Fig. 6B (lane 3) clearly demonstrated that
HNF4
interacts with TR4 in vitro. However, the
interaction of the deletion mutants of GST-TR4 fusion protein with
HNF4
is much weaker, suggesting that instead of direct competition
for binding to the TR4RE-HBV, TR4 may interact with HNF4
, which
could possibly result in the repression of HNF4
function. To further
confirm this interaction in liver cells, we co-transfected TR4 and
HNF4
into COS1 cells and used a co-immunoprecipitation assay to
immunoprecipitate the TR4 and HNF4
complex by an anti-TR4 monoclonal
antibody. As shown in Fig. 6C (lane 4), we can
clearly observe HNF4
bands in the precipitated complex by Western
blotting when co-expressing TR4 and HNF4
proteins in COS1 cells.
This result suggests that TR4 can bind to HNF4
.
transcriptional activity by interacting with
HNF4
. Also, the full-length of TR4 is essential for this regulation.
We further compared the effects of TR4 on HNF4
-mediated transactivation on the CpLUC reporter with deletion mutants of TR4 in
HepG2 cells using transient transfection assay (Fig. 6D). As
expected and similar to the result shown in Fig. 4A,
TR4 suppresses HNF4
-mediated transactivation (Fig. 6D,
column 3 versus 4). However, the N- or
C-terminal deletion of TR4 did not exhibit similarly suppressive
effects as those of the full-length TR4 did (column 4 versus 7 and 8), implying that these
two domains are essential in the protein-protein interaction of TR4 and
HNF4
, and this interaction is required for the repressive action of
TR4 on the transcriptional activity of HNF4
.
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Fig. 7.
Primer extension analysis of the effect of
TR4 on HBV pre-core and core RNA expression. HBV RNA isolated from
Huh-7 cells transfected with HBV DNA and TR4 or the parental vector of
TR4 as indicated was analyzed by primer extension assay as described
under "Experimental Procedures." Briefly, the products of the
primer extension were subjected to electrophoresis on an 8% sequencing
gel. The locations of the core, pre-core, and human growth hormone
(hGH) RNA are indicated. The hGH served as an internal
control. The results were visualized by autoradiography and quantified
by Sigma scan software. The value of the quantification represents
three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, COUP-TF1, and RXR
-PPAR
(37). The other
site (AGGTTAAAGGTCT, nucleotide coordinates 1757-1769) is located in the core promoter that can be recognized by
HNF4
and TR4 as described in current studies (13). The detailed mechanisms of how these nuclear receptors modulate HBV gene expression via these two DR1 sites remain unclear. Our data strongly suggest that
TR4 preferentially binds the DR1 site of the core promoter, because TR4
suppression is abrogated if this site is mutated (Fig. 4B).
The selective binding of TR4 to the 2nd DR1 site may be a key step for
TR4 to prevent other nuclear receptors from binding to this site,
thereby repressing transcription. However, we cannot rule out the
possibility that TR4 may also regulate HBV gene expression through the
DR1 site of enhancer I, because our data indicate that TR4 can
recognize this site (data not shown). Moreover, because TR4 binding to
the 2nd DR1 site is prevented by two mutations ((1756 (A to T) and 1767 (G to A)), which are frequently found in patients with chronic
hepatitis, one may speculate that HBV may utilize these two mutations
to escape suppression mediated by TR4 and/or other transcriptional
repressors. Alternatively, creation of new mutations at these consensus
DR1 sites may be able to increase the opportunity for other modulators
to regulate HBV expression via these mutated DR1 sites (32).
suggests that the
TR4 affects HBV gene expression at multiple levels, most importantly, including the DNA binding and protein-protein interactions.
Nevertheless, previous reports also suggested that COUP-TF could
differentially modulate target gene expression via similar HREs. For
example, COUP-TF1 can induce the 7
-hydroxylase promoter activity via
the DR4 binding site and repress myosin heavy chain promoter activity via a similar DR4 binding site (39). Similar differential modulation of
target gene expression also occurred with TR4, via binding to similar
DR1 binding sites (21, 23).
(DR1), VDR
(DR3), and retinoic acid receptor
(DR5), for the same consensus HRE
sites (21, 22). Previous studies showed that ligand-activated RXR
/PPAR
or PPAR
can increase HBV core promoter activity
through the TR4RE-HBV site (DR1) (13, 14). Therefore, it appears that TR4 may compete for the same binding site with these nuclear receptors resulting in repression of HBV core promoter activity, which this regulation does not involve in any interaction between TR4 and other
nuclear receptors (21, 22). Second, TR4 can also modulate gene
expression via protein-protein interactions with other nuclear receptors. For example, TR4 can suppress androgen target genes through
interactions with the androgen receptor (40). In the current study, we
found TR4 cannot only bind to the same DR1 site on HBV core promoter as
HNF4
(Fig. 5), but also interact with HNF4
(Fig. 6, B
and C). The TR4-mediated repression resulted from this
interaction was confirmed by using TR4 deletion mutants in several
parallel experiments and may further support that there might be
multiple mechanisms by which TR4 proceeds in this repressive regulation. Previous studies showed that TR2, a close member of TR4
family, and RIP140 (receptor-interacting protein 140), one of
TR4-interacting proteins, can interact and recruit histone deacetylases
for gene silencing (22, 41, 42). It is possible that TR4 may carry out
this repression through similar mechanisms, which would be an
interesting direction for our future study to test the involvement of
histone deacetylases in this regulation.
could be a unique way to relieve TR4-mediated repression,
because HNF
can reduce TR4 binding to the DR1 site through either
competition for DNA binding or protein-protein interactions (Fig. 5).
The underlying mechanisms of how these nuclear receptors differentially
influence the transcription activation of HBV genes during infection
will require more extensive study.
, to form an inactive protein
complex. Either of these two activities may result in the suppression
of HBV core promoter.
View larger version (18K):
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Fig. 8.
Schematic diagram of the regulation of the
HBV core promoter by TR4 through the DR1 (TR4RE-HBV) site. This
figure depicts a simple model of the HBV core promoter and the
regulation of this promoter via TR4RE-HBV by TR4. A, The
binding of TR4 to the TR4RE may restrict the accessibility of this site
to other essential nuclear receptors for transcriptional activation.
Also, the interaction of HNF4 and TR4 inhibits HNF4
transcriptional activity and results in the suppression of the HBV core
promoter activity. B, during natural infection, by an
unknown mechanism, the interaction of HNF4
and 2R4 may prevent TR4
from binding to the DR1 site (Fig. 5). Therefore, this regulation can
relieve TR4-mediated repression and allow HNF4
or other nuclear
receptors to proceed to the transcriptional activation.
In conclusion, this study provides evidence that TR4 is a
transcriptional repressor of the HBV core promoter. The interaction of
TR4 and HNF4 further extends the scope of nuclear receptor regulation of the HBV core promoter. The transcriptional activation of
the core promoter is a key step to initiate the life cycle of HBV
during the infection (34). By identifying of physiological ligand(s)
for TR4, we may be able to control agonist or antagonist binding to TR4
to modulate HBV gene expression, which may provide a novel strategy to
develop anti-HBV drugs.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Alan McLachlan for the HBV
reporter plasmids, Dr. Margarita Hadzopoulou-Cladaras for the HNF4
plasmid, and Dr. Kung-Liang Guan for the pG4LUC. We are grateful to
Karen Wolf and Erik R. Sampson for the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK56984 and DK47258 (to C. C.) and CA77817 (to J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: George Whipple Lab
for Cancer Research, Departments of Pathology, Urology, and Radiation
Oncology, and the Cancer Center, University of Rochester Medical
Center, 601 Elmwood Ave., Box 626, Rochester, NY 14642. Tel.:
585-273-4500; Fax: 585-756-4133; E-mail:
chang@urmc.rochester.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M205944200
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ABBREVIATIONS |
---|
The abbreviations used are:
HBV, hepatitis B
virus;
TR4, testicular receptor 4;
HNF4, hepatocyte nuclear factor 4
;
DR, direct repeat;
RXR, retinoid X receptor;
PPAR
, peroxisome
proliferator-activated receptor
;
TR2, testicular receptor 2;
C
mRNA, core mRNA;
pre-C mRNA, pre-core mRNA;
C, core;
HRE, hormone
response element;
COUP-TF1, chicken ovalbumin upstream
promoter-transcription factor 1;
VDR, vitamin D receptor;
DBD, DNA-binding domain;
LBD, ligand-binding domain;
EMSA, electrophoretic
mobility shift assay;
GST, glutathione S-transferase.
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