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J Biol Chem, Vol. 274, Issue 5, 2858-2865, January 29, 1999
From the Department of Molecular Biology and the Research Center
for Cell Differentiation, Seoul National University,
Seoul 151-742, Korea
The hepatitis B viral X protein (HBx) is known to
exert its transactivation activity by the interaction with several
cellular transcription factors. Here we report the interaction of HBx
and CCAAT/enhancer-binding protein Hepatitis B virus (HBV)1
is a partially double-stranded DNA virus that replicates through the
reverse transcription of pregenomic RNA (1). HBV is a causative agent
of chronic and acute hepatitis and is associated with the development
of hepatocellular carcinoma (2). The X protein of HBV (HBx) has been
implicated in HBV-mediated hepatocellular carcinoma by its abilities to
induce liver cancer in some transgenic mice (3) and to transactivate a
variety of viral and cellular promoters (reviewed in Ref. 4). Being unable to bind DNA directly, the activity of HBx is known to be mainly
mediated through the binding sites for other transcription factors such
as AP-1 (5), NF- HBx has been shown to interact with some basal transcription factors
(13-17), the p53 tumor suppressor (18, 19), and basic leucine zipper
(bZip) proteins (8, 20). Stimulation of RNA polymerase II transcription
by HBx in vitro or in cultured cells seems to require a
specific cis-element or other activator proteins (9, 14,
16), which suggests a role for HBx as a coactivator. These two distinct
roles for HBx, as a cellular signaling molecule or as a coactivator of
transcription, have been suggested to be dependent on its intracellular
distribution (21). This is also implied for the interaction of HBx and
the p53 tumor suppressor, which modulates p53-mediated gene
transcription and apoptosis (18, 19, 22). Other functions of HBx
suggested so far include the interactions of HBx with UV-damaged
DNA-binding protein (23), proteasome complexes (24), and protease
tryptase TL2 (25). Recent reports suggesting a role of HBx
in DNA repair are noteworthy (17, 26). Two distinct functions and these
alternatives of HBx action may explain the multifunctional role of HBx
in vivo.
Although the role of HBx during the infection of HBV is still unclear,
it seems to play an essential role in infection by woodchuck hepatitis
B virus (27, 28). In addition, transactivation of HBV
enhancer/promoters by HBx (29, 30) increases its importance in the HBV
life cycle. A binding site for AP-1, a ubiquitous transcription factor,
in enhancer I is the only cis-element of HBV yet known to
respond functionally to HBx (7).
The CCAAT/enhancer-binding protein (C/EBP) was first identified in rat
liver (31) and is expressed mainly in highly differentiated cells such
as liver and fat cells (32), where it plays a key role in cell
differentiation (33, 34). C/EBP belongs to the bZip family of
transcription factors and activates transcription of several genes
through its binding sites (a consensus site: 5'-RTTGCGYAAY-3') (35) in
liver and fat cells. C/EBP has been shown to bind and modulate enhancer
I (36) and the enhancer II/core promoter (37, 38) of HBV. A possible
role of C/EBP in the HBx-stimulated expression of promoters other than
HBV has been previously suggested by indirect evidence (39, 40). In the
course of our study, a physical interaction of HBx and C/EBP Plasmid Construction--
The pCENCAT reporter plasmid was
constructed by inserting the XhoI-AluI fragment
(nt 127-1873) of HBV subtype adr-k (41), which contains the
upstream enhancer, the enhancer I/X promoter (EnI/Xp), and EnII/Cp, in
front of the CAT reporter gene (see Fig. 1A). Plasmids
pUEIXp and pCpBm were constructed by dividing the HBV
enhancer/promoters using the BamHI site (nt 1937) into the
regions containing the upstream enhancer and EnI/Xp, and EnII/Cp, respectively. The pEIXp plasmid was constructed by deleting the XhoI-AccI fragment (nt 127-1070) containing the
upstream enhancer from pUEIXp. The derivatives of pCpBm were
constructed by serially digesting the plasmid with SacII
(pCpSc), ApaLI (pCpAp), StyI (pCpSy),
HincII (pCpHc), and DraI (pCpDa) as described
previously (42). The position of each deletion site is indicated in
Fig. 2A. The pC/EBP-CAT reporter plasmid was constructed by
inserting eight copies of the consensus C/EBP-binding site
(5'-TGCAGATTGCGCAATCTGCA-3') into the BglII site of the
pCATpromoter (Promega) containing the minimal SV40 early promoter. The
pBLCAT2 plasmid has a minimal promoter (
The C/EBP Transient Transfection and CAT Assay--
HepG2 cells were
transfected with reporter and activator plasmids as indicated in the
figure legends using the calcium phosphate co-precipitation method with
BES as described previously (7). The total amount of transfected DNA
for each reaction was adjusted to 9 µg with pUC19. The CAT assay was
performed with cell extracts normalized for the total amount of protein
using the Bradford assay (Bio-Rad). CAT activity was quantified by
measuring the conversion of [14C]chloramphenicol to its
acetylated forms using a Fuji BAS bioimaging analyzer.
Preparation of Recombinant Proteins and Electrophoretic Mobility
Shift Assay (EMSA)--
The GST-fused C/EBP
Antibody against human C/EBP In Vitro Interaction Assay--
One milliliter of bacterial
extracts containing GST-C/EBP HBx and C/EBP Identification of the Region in HBV EnII/Cp Responsible for the
Synergism between HBx and C/EBP
To find the binding site of C/EBP Synergistic Effect of HBx and C/EBP HBx Binds to the bZip Domain of C/EBP Central Region of HBx Is Necessary for the Direct Interaction with
C/EBP
EMSA and the CAT assay using HBx derivatives showed somewhat different
results. MBP-middle X protein did not efficiently enhance the binding
of C/EBP We have demonstrated here that HBx enhanced the binding activity
of C/EBP The synergistic effect of HBx and C/EBP Serial deletion analysis of the HBV pregenomic promoter and EMSA
revealed the region (nt 1639-1679) with two C/EBP-binding sites
functionally responsible for the effect of C/EBP The C/EBP family proteins were reported to interact directly and/or
functionally with various transcription factors, including HNF-1 (58),
v-Myb (59), AML1 (60), glucocorticoid receptor (61), HNF-4 (57), Sp1
(56), and NF- HBx is somewhat different from other C/EBP-interacting proteins in its
inability to bind DNA. The role of HBx in the interaction with C/EBP Domain analysis tentatively revealed that the C-terminal two-thirds of
HBx are important in the direct interaction with the bZip domain of
C/EBP We thank Dr. S. L. McKnight for the kind
gift of the C/EBP *
This work was supported in part by the Korea Ministry of
Education and the Korea Science and Engineering Foundation through the
Research Center for Cell Differentiation at Seoul National University.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.
The abbreviations used are:
HBV, hepatitis B
virus; HBx, hepatitis B viral X protein; CREB, cAMP-responsive
element-binding protein; bZip, basic leucine zipper; C/EBP, CCAAT/enhancer-binding protein; EnII/Cp, enhancer II/pregenomic
promoter; EnI/Xp, enhancer I/X promoter; nt, nucleotide(s); CAT, chloramphenicol acetyltransferase; GST, glutathione
S-transferase; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; EMSA, electrophoretic mobility shift assay; MBP, maltose-binding protein.
Interaction of Hepatitis B Viral X Protein and
CCAAT/ Enhancer-binding Protein 
Synergistically Activates
the Hepatitis B Viral Enhancer II/Pregenomic Promoter*
ABSTRACT
Top
Abstract
Introduction
References
(C/EBP) and their effects on the enhancer/promoters of hepatitis B virus (HBV). A chloramphenicol acetyltransferase assay showed that the cotransfection of HBx and
C/EBP strongly activated the enhancer II/pregenomic promoter of HBV
in a synergistic manner. This effect was also observed in the
heterologous expression system with promoters of SV40 and herpes
simplex virus thymidine kinase genes. Serial deletion analysis of the
enhancer II/pregenomic promoter identified the responsible region
(nucleotides 1639-1679), in which two C/EBP-binding sites are located.
An in vitro interaction assay and electrophoretic mobility
shift assay showed that HBx augmented the DNA binding activity of
C/EBP by direct interaction with it, and its basic leucine zipper
domain was responsible for the interaction with HBx. Domain analysis of
HBx showed that the central region (amino acids 78-103) was necessary
for direct interaction with C/EBP. However, the complete form of HBx
was necessary for the synergistic activation of the HBV pregenomic
promoter. These results suggest that the interaction of HBx and
C/EBP enhances the transcription of the HBV pregenomic promoter for
the effective life cycle of HBV in hepatocytes.
INTRODUCTION
Top
Abstract
Introduction
References
B (6), ATF/CREB (7, 8), and acidic activators (9).
HBx has been shown to activate AP-1 and NF-B by means of
Ras-mediated signaling pathways (5, 6, 10). Activation of Ras by HBx
was also shown to increase the cellular level of TATA-binding protein,
which induces RNA polymerase III-dependent transcription
(11). Recently, it was reported that activation of Src family kinases
by HBx is coupled with the activation of Ras (12).
without
any functional implications in vivo was reported (20). We
investigated a role for HBx in C/EBP function and their effects on
the HBV enhancer/promoters in HepG2 cells. We report here that the
direct interaction of HBx and C/EBP strongly activated the enhancer
II/pregenomic promoter (EnII/Cp) of HBV in a synergistic manner.
EXPERIMENTAL PROCEDURES
150 to +50) of the herpes
simplex virus thymidine kinase gene that contains its own C/EBP-binding
site (43).
expression vector pMSV-C/EBP is a kind gift of Dr.
S. L. McKnight. The bacterial expression vectors for full-length C/EBP and the bZip domain of C/EBP (pGST-C/EBP and pGST-bZip, respectively) were constructed by inserting the
NcoI-NcoI fragment (amino acids 1-358) and the
SmaI-NcoI fragment (amino acids 245-358) of
C/EBP cDNA into the SmaI site of pGEX-4T1 and
pGEX-4T2 (Amersham Pharmacia Biotech), respectively. The eukaryotic
expression vector for HBx (pSVX2) was constructed by placing its
cDNA (nt 1371-1833) under the control of the SV40 enhancer and
early promoter. The derivatives of pSVX2 (pSVX
BA, pSVXBH, and
pSVXSH) were constructed using the BamHI,
ApaLI, and HincII sites, respectively, within the
HBx cDNA as described for Fig. 5A. The bacterial
expression vectors for HBx and its derivatives (pMBP-X, pMBP-Mx, and
pMBP-Sx) were described previously (7).
and MBP-fused X
proteins were affinity-purified, and the amount of purified proteins
was determined by the Bradford assay. DNA binding reactions were
carried out with the indicated amount of proteins as described
previously (7), except that the proteins were preincubated in a
reaction mixture without a probe for 10 min at room temperature and
then incubated with labeled probes for 5 min. Samples were loaded on a
5% native polyacrylamide gel (40:1 acrylamide/bisacrylamide) in 0.5×
Tris borate/EDTA, and the gels were dried and exposed to x-ray film.
was from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The sequences of the probes covering the HBV
enhancer/promoters are as follow: nt 974-995,
5-TATTGACTGGAAAGTATGTCAA-3'; nt 1024-1046,
5'-CCTTTTACACAATGTGGCTATCC-3'; nt 1170-1198,
5'-CTGCCAAGTATTTGCTGACGCAACCCCCA-3'; nt 1639-1673,
5'-CCAAGGTCTTACATAAGAGGACTCTTGGACTCTCA-3'; nt 1666-1690, 5'-GACTCTCAGCAATGTCAACGTCCGA-3'; nt 1680-1706,
5'-TCAACGTCCGACCTTGAGGCATACTTC-3'; nt 1699-1729,
5'-CATACTTCAAAGACTGTTTGTTTAAAGACTG-3'; nt 1725-1751, 5'-GACTGGGAGGAGTTGGGGGAGGAGATT-3'; nt 1740-1761,
5'-GGGGAGGAGATTAGGTTAATGA-3'; nt 1798-1822,
5'-GTTCACCAGCACCATGCAACTTTTT-3'; and nt 1826-1846, 5'-CCTCTGCCTAATCATCTCATG-3'. Sequences homologous to the
consensus C/EBP site are underlined. The sequences of the wild-type and mutant C/EBP-binding sites are as follow: C/EBPwt,
5'-TGCAGATTGCGCAATCTGCA-3'; and C/EBPmt,
5'-TGCAGAGACTAGTCTCTGCA-3'. The consensus C/EBP site in
C/EBPwt and the mutated sequences in C/EBPmt are underlined.
or GST-bZip were incubated with 20 µl of amylose resin bound to MBP or MBP-fused HBx proteins for
12 h at 4 °C. After extensive washing (10 × 10-min
incubation with 1 ml of column buffer (10 mM sodium
phosphate (pH 7.2), 0.5 M NaCl, 1 mM sodium
azide, 1 mM EGTA, and 10% glycerol)), bound proteins were
eluted with 50 µl of column buffer containing 10 mM
maltose. Ten microliters of eluted proteins were assayed by
SDS-polyacrylamide gel electrophoresis and electrophoretically
transferred to nitrocellulose membranes at 4 °C. Membranes were
blocked with 5% nonfat dry milk in phosphate buffer (80 mM
Na2HPO4, 20 mM
NaH2PO4 (pH 7.5), 100 mM NaCl, and 0.1% Triton X-100), briefly washed, and incubated with anti-C/EBP antibody (1:5000) for 1 h at 25 °C. After extensive washing,
membranes were incubated with a horseradish peroxidase-linked
anti-rabbit mouse antibody (1:10,000) for 1 h at 25 °C.
Protein-antibody complexes were visualized by the ECL Western blotting
detection system (Amersham Pharmacia Biotech) according to the
manufacturer's instructions.
RESULTS
Synergistically Activate the Pregenomic Promoter
of HBV--
HBV has been previously shown to have C/EBP-binding sites
in the upstream enhancer, enhancer I , and EnII/Cp (37, 44). We first
examined the effect of HBx and C/EBP on the whole HBV enhancer/promoters (pCENCAT) in HepG2 cells, which contain low levels
of C/EBP compared with normal liver (45). The effects of both
proteins on each enhancer/promoter (pCpBm, pUEIXp and pEIXp) were then
examined. As shown in Fig. 1B
(lanes 3, 7, 11, and
15), HBx alone activated all four reporters. In contrast to previous reports (36, 38), C/EBP alone neither activated nor
repressed notably the HBV enhancer/promoters in our experiments (Fig.
1B, lanes 2, 6,
10, and 14). However, cotransfection of HBx and
C/EBP significantly elevated the activities of pCENCAT and pCpBm
plasmids (Fig. 1B, lanes 4 and
8), suggesting a synergistic effect of HBx and C/EBP on
HBV EnII/Cp. Contrary to our expectation, the cotransfection of HBx and
C/EBP had only a small effect on pUEIXp (Fig. 1B,
lane 12) and inhibited the activation of pEIXp by HBx
(lane 16), although they have C/EBP-binding sites. By EMSA, purified C/EBP efficiently bound to the known C/EBP site of enhancer I, but weakly bound and did not bind to the downstream and upstream C/EBP sites of the upstream enhancer, respectively (data not shown). When compared with the consensus sequence of C/EBP, the C/EBP site at
the upstream site of the upstream enhancer (nt 974-995) does not have
any homology, whereas the one at the downstream site (nt 1024-1046)
and the other one in enhancer I (nt 1170-1198) have 80% homology
(Table I). The discrepancy of the results
from the CAT assay and EMSA indicates that C/EBP did not act as an influential activator in EnI/Xp.
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Fig. 1.
Synergistic effect of HBx and
C/EBP on the HBV enhancer/promoters.
A, schematic presentation of HBV enhancer/promoter
constructs. Known factor-binding sites are shown at the top. As
indicated, plasmid pCENCAT has a whole region from the upstream
enhancer (UE) to enhancer II (EnII) and the
pregenomic promoter (Cp) (nt 127-1873 of HBV subtype
adr-k). Plasmids pCpBm and pUEIXp have the regions
encompassing the pregenomic promoter and the X promoter
(Xp), respectively, of pCENCAT. Plasmid pEIXp has EnI/Xp
alone without the upstream enhancer. Ac, AccI;
Bm, BamHI. B, 3 µg of pUEIXp and 1 µg each of the other reporters were transfected into HepG2 cells with
the HBx (pSVX2) and C/EBP (pMSV-C/EBP) expression vectors as
indicated. pMSV-C/EBP (0.5 µg) and pSVX2 (4 µg) were used for
each reporter plasmid.
Sequence comparison of the consensus C/EBP site in the HBV probes by
EMSA in this study
--
We analyzed further the
EnII/Cp region that was highly activated by the cotransfection of HBx
and C/EBP (Fig. 1B). Previously, HBV EnII/Cp has been
shown to contain several C/EBP-binding sites (37). To identify the
responsible one in EnII/Cp, the effect of HBx and C/EBP on the
serial deletions of pCpBm was examined by CAT assay (Fig.
2A). The basal activities of
plasmids pCpAp and pCpDa were lower than those of the other reporter
plasmids, which is consistent with previous reports (42, 46). The
synergistic effect of HBx and C/EBP was maintained on plasmid pCpSy,
but not on plasmids pCpHc and pCpDa. In contrast, HBx alone slightly activated pCpHc. These results show that the
StyI-HincII fragment (nt 1639-1679) in the
enhancer II region is responsible for the synergistic effect of HBx and
C/EBP on the HBV pregenomic promoter.
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Fig. 2.
Identification of the region in EnII/Cp
responsible for the synergistic effect of HBx and
C/EBP . A, serial deletion
analysis of EnII/Cp. Serial deletion clones of pCpBm were made using
the restriction enzymes indicated at the top and were examined for the
effect of HBx and C/EBP using a CAT assay. Known C/EBP-binding sites
of EnII/Cp are indicated by closed circles and are
numbered consecutively. One microgram each of the deletion
clones was transfected into HepG2 cells in combination with 4 µg of
pSVX2 and 0.5 µg of pMSV-C/EBP as described in the legend to Fig.
1. Relative CAT activities are listed on the right. Bm,
BamHI; Sa, SacII; Ap,
ApaLI; Sy, StyI; Hc,
HincII; Da, DraI. B,
identification of the binding site of C/EBP in HBV EnII/Cp.
Oligonucleotides covering sequences downstream of the StyI
site (nt 1639) were used as probes for EMSA as indicated, and their
sequences are described under "Experimental Procedures." A
consensus C/EBP-binding site was used as a positive control. EMSA was
accomplished with 50 ng of GST-C/EBP as described under
"Experimental Procedures." The DNA-protein complex is indicated by
the arrow. C, HBx-enhanced binding of C/EBP to
HBV EnII/Cp and competition analysis. EMSA was carried out on the
oligonucleotides at nt 1639-1673 (lanes 1-5) and nt
1666-1690 (lanes 6-10) with 50 ng of GST-C/EBP and 100 ng of MBP-X protein as indicated. Purified MBP was added to the
reaction mixtures without MBP-X protein to adjust the amount of total
proteins (lanes 1 and 6). A 50-fold molar excess
of the unlabeled probes (self) and the consensus (C/EBPwt)
and mutant C/EBP (C/EBPmt) oligonucleotides were added as competitors
(lanes 3-5 and 8-10). The sequences of C/EBPwt
and C/EBPmt are also described under "Experimental
Procedures."
in HBV EnII/Cp, we performed EMSA
with oligonucleotides synthesized based on the sequences of the
probable C/EBP sites and the previous data on this region (37, 47, 48).
Purified GST-C/EBP bound to the regions spanning nt 1639-1673 and
1666-1690 (Fig. 2B, lanes 1 and
2; known C/EBP sites depicted as sites 1 and
2 in Fig. 2A), but not to the other regions (Fig.
2B, lanes 3-8; downstream of the
HincII site). The consensus C/EBP site (C/EBPwt), used as a
positive control, also bound to GST-C/EBP (Fig. 2B,
lane 9). Siddiqui and co-workers (37) have identified the
multiple C/EBP-binding sites in this region and the downstream
sequences near the translation start site of core gene expression by
DNase I protection analysis. However, downstream C/EBP sites (nt
1740-1761, 1798-1822, and 1826-1846; known C/EBP sites depicted as
sites 3-5 in Fig. 2A) were not bound by purified
C/EBP in our experiment (Fig. 2B, lanes
6-8), although all these sequences have 60-70% homologies
to the consensus C/EBP site (Table I). The discrepancy between our
results and theirs may be due to the assay methods, and C/EBP
binding to DNA seems to require conservation of particular bases or
secondary structure more than a consensus sequence (35, 49). The
binding of GST-C/EBP to these two regions (nt 1639-1673 and
1666-1690) was augmented by the addition of purified MBP-X protein
(Fig. 2C, lanes 2 and 7)
without a supershifted band, which is consistent with previous reports
(8, 20). This binding was abolished by the unlabeled probe and
consensus C/EBP oligonucleotide (Fig. 2C, lanes
3, 4, 8, and 9), but not by the mutant C/EBP
oligonucleotide (lanes 5 and 10). These results
show that HBx enhances the binding of purified C/EBP to the two
C/EBP-binding sites in nt 1639-1690 of HBV enhancer II, which is
consistent with the results of the CAT assay (Fig. 2A).
on the Heterologous
Promoters--
The synergistic effect of HBx and C/EBP was tested
first on the minimal SV40 promoter with or without C/EBP-binding sites (Fig. 3A). As shown in Fig.
3B, both reporter plasmids were activated by HBx
(lanes 3 and 7), but the synergistic
effect of HBx and C/EBP was shown only with pC/EBP-CAT, which
suggests a role of the C/EBP-binding site (compare lanes
4 and 8). C/EBP alone slightly enhanced the
activity of pC/EBP-CAT (Fig. 3B, lane 6). The
effect of HBx and C/EBP was then examined on the thymidine kinase
gene promoter of herpes simplex virus, which has its own C/EBP-binding site located ~80 base pairs upstream of the transcription start site
(43). As reported previously (50), C/EBP alone highly activated the
thymidine kinase promoter (Fig. 3C, lane 2). HBx alone slightly activated the thymidine kinase promoter (Fig.
3C, lane 3), but activated it >40-fold in the
presence of C/EBP (lane 4). The synergistic effect on the
thymidine kinase promoter was stronger than that on HBV EnII/Cp and the
SV40 promoter and was effective even with the low concentration of HBx
and C/EBP (data not shown). These results show that the cooperation
of HBx and C/EBP is also shown on the other promoters depending on
the C/EBP-binding site.
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Fig. 3.
Synergistic effect of HBx and
C/EBP on the minimal promoters of SV40 and
thymidine kinase genes. A, schematic presentation of
reporters. The plasmid pCATpromoter has a minimal SV40 early promoter
(SV40-p) without a C/EBP-binding site. The plasmid
pC/EBP-CAT has eight copies of the consensus C/EBP-binding site before
the SV40 promoter of the pCATpromoter. The plasmid pBLCAT2 has a
minimal thymidine kinase gene promoter (tk-p) with its own
C/EBP-binding site. B, the role of the C/EBP-binding site in
the activation of the minimal SV40 promoter by HBx and C/EBP. Three
micrograms of each reporter, 4 µg of pSVX2 (HBx), and 0.5 µg of pMSV-C/EBP (C/EBP) were transfected
into HepG2 cells as indicated. C, effect of HBx and C/EBP
on the minimal promoter of the thymidine kinase gene. Two micrograms of
pBLCAT2 were transfected into HepG2 cells with 2 µg of pSVX2
(HBx) and 0.5 µg of pMSV-C/EBP
(C/EBP).
--
We analyzed the
direct interaction of HBx and full-length C/EBP or the bZip domain
of C/EBP using an in vitro interaction assay with MBP-X
protein-coupled amylose resin (Fig.
4A). Both full-length C/EBP
and the bZip domain of C/EBP (GST-C/EBP and GST-bZip,
respectively) directly bound to MBP-X protein (Fig. 4A,
lanes 2 and 4), but not to MBP alone
(lanes 1 and 3). Purified GST-C/EBP
and GST-bZip proteins were used as markers (Fig. 4A, lanes M1 and M2). MBP-X protein did
not bind to GST protein (data not shown). The effect of HBx on the DNA
binding affinity of C/EBP was then examined by EMSA of the consensus
C/EBP-binding site (the C/EBPwt probe). As shown in Fig. 4B
(lanes 2 and 7), MBP-X protein
increased the binding of both GST-C/EBP and GST-bZip to the
consensus C/EBP site. The addition of anti-C/EBP antibody produced
supershifted bands (Fig. 4B, lanes 3 and 8), and the complexes disappeared with the C/EBPwt
competitor (lanes 4 and 9), but not
with the mutant competitor (C/EBPmt) (lanes 5 and 10). These results show that HBx targets the bZip domain of
C/EBP and enhances its binding to DNA.
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Fig. 4.
HBx directly interacts with the bZip domain
of C/EBP and enhances its binding to the
consensus C/EBP site. A, in vitro
interaction assay of the direct interaction of HBx and C/EBP as
described under "Experimental Procedures." MBP or MBP-X protein was
used as a bait for full-length C/EBP (lanes 1 and 2) or the bZip domain of C/EBP (lanes 3 and 4). Eluted proteins were subjected to Western blotting
using anti-C/EBP antibody. Purified GST-C/EBP and GST-bZip were
used as markers (lanes M1 and M2, respectively).
B, HBx-enhanced binding of C/EBP to the consensus C/EBP
site. Fifty nanograms of full-length C/EBP (GST-C/EBP) or the
bZip domain of C/EBP (GST-bZip) were incubated with the
32P-labeled consensus C/EBP oligonucleotide (C/EBPwt) in
the presence (lanes 2-5 and 7-10, respectively)
or absence (lanes 1 and 6) of 100 ng of MBP-X
protein. Anti-C/EBP antibody (Ab) was added just before
the addition of the probe (lanes 3 and
8). A 50-fold molar excess of unlabeled C/EBPwt (lanes
4 and 9) or C/EBPmt (lanes 5 and
10) was used as a competitor.
--
To assign the domains of HBx that are important for the
interaction with C/EBP, derivatives of HBx were made (Fig.
5A). N-terminal deletions of
HBx were made based on the two internal ATG codons, and the C-terminal
deletion clone lacks its acidic transactivation domain. Smaller forms
of HBx have been reported to be expressed from an intragenic promoter
(51, 52) and to transactivate differentially several class II promoters
(53). Direct interaction of C/EBP and the HBx derivatives (MBP-X
protein, MBP-middle X protein, and MBP-small X protein) was examined by
in vitro interaction assay as described above (Fig.
5B). Full-length and middle X proteins bound directly to
both GST-C/EBP (Fig. 5B, lanes 2 and 3) and GST-bZip (lanes 6 and
7), but small X protein (lanes 4 and
8) and MBP alone (lanes 1 and
5) did not. Purified GST-C/EBP and GST-bZip proteins were
used as markers (Fig. 5B, lanes M1 and M2). Coomassie Blue staining with an equal volume of
bacterial extracts showed that the result was not due to the
differences in the amounts of HBx proteins expressed in
Escherichia coli (data not shown). These results suggest
that the central region of HBx is necessary for the direct interaction
with C/EBP.
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Fig. 5.
Domain analysis of HBx. A,
shown is a schematic presentation of full-length HBx and its deletion
mutants. The HBx derivatives used for the mammalian and bacterial
expression are described under "Experimental Procedures."
HBx, full-length X protein; Mx, middle X protein;
Sx, small X protein; C, C-terminal deletion
mutant of HBx. B, the direct interaction of C/EBP and HBx
derivatives was subjected to in vitro interaction assay.
Full-length C/EBP (lanes 1-4) or the bZip domain of
C/EBP (lanes 5-8) was assayed as described in the legend
to Fig. 4. Purified GST-C/EBP and GST-bZip were used as markers
(lanes M1 and M2, respectively). C, 50 ng of GST-C/EBP were incubated with 100 ng each of the purified HBx
derivatives (MBP-X protein (MBP-X), MBP-middle X protein
(MBP-Mx), and MBP-small X protein (MBP-Sx)). EMSA
was accomplished as described under "Experimental Procedures."
D, 4 µg each of the HBx derivatives (pSVX2, pSVXBA,
pSVXBH, and pSVXSH) and 0.5 µg of pMSV-C/EBP were
transfected into HepG2 cells as indicated, and the CAT assay was
performed.
to the C/EBP site in EMSA (Fig. 5C, compare lanes 2 and 3). As expected, MBP-small
X protein showed no effect (Fig. 5C, lane 4). The
results of the CAT assay using pC/EBP-CAT as a reporter also showed
that smaller forms of HBx did not synergize with C/EBP as
efficiently as full-length X protein (Fig. 5D, compare
lane 2 with lanes 3-5). In contrast
to full-length HBx (Fig. 3B, lane 7), smaller
forms of HBx by themselves did not activate pC/EBP-CAT (data not
shown). These results suggest that the functional synergism of HBx and
C/EBP requires more than direct interaction of both proteins. The
whole region of HBx is likely to be needed.
DISCUSSION
on the DNA by directly interacting with its bZip domain and
that the interaction of both proteins synergistically transactivated
the pregenomic promoter of HBV. These results revealed the C/EBP as
a cellular target of HBx for the transactivation of the HBV pregenomic
promoter and can accordingly explain its liver specificity. The role of
C/EBP family proteins in the function of HBx has been suggested
previously by indirect evidence (39, 40), and recently, the -isoform
of C/EBP has been reported to interact with HBx without any effect
on the functional implications in vivo or in cultured cells
(20). However, our results clearly showed the functional synergism and
direct interaction of HBx and C/EBP both in HepG2 cells and in
vitro.
differentially regulated
EnI/Xp and EnII/Cp of HBV, although both promoters have C/EBP-binding
sites (Fig. 1). This result is also implied by the effect on the
heterologous promoters of SV40 and thymidine kinase genes (Fig. 3) and
is relevant to a previous report that showed the differential
regulation by C/EBP of viral enhancer/promoters including HBV
enhancer I depending on the promoter context (50). Activation of its
own promoter (X promoter) by HBx seems to be mainly mediated by the
AP-1-binding site in HBV enhancer I (7).
and HBx (Fig. 2).
This region has also been shown to bind other transcription factors
(54, 55), of which HNF-4 and Sp1 were shown to synergize with C/EBP
(56, 57). Other known C/EBP-binding sites of HBV EnII/Cp also have
homologies to the consensus C/EBP site similar to these sites, but did
not bind to purified C/EBP in our experiment (Fig. 2B).
Extensive analysis of the binding sites of C/EBP has shown that
bases at ±3 and ±4 in the 10-base pair consensus sequence are most
important and that bases at ±1 and ±5 are least important (35, 49).
In addition, the consensus sequence of the half-site 5'-RTTGC-3'
appears to be more important for C/EBP binding than the palindrome
structure of DNA sequences. The C/EBP sequences in the regions
spanning nt 1639-1673 and 1666-1690 are more similar to the above
requirements than other sites (Table I).
B (62-64), which synergistically activates the
responsive promoters through their overlapping binding sites. Among
these factors, Sp1 synergizes with C/EBP, but not with C/EBP
(56), and the interactions of NF-B with C/EBPs are so complex that
p50 and p65 subunits of NF-B seem to selectively interact with the
different isoforms of C/EBP to activate or repress promoters depending
on the cis-elements. From these facts, it is likely that the
C/EBP family proteins can interact with HBx differentially to act on
the different enhancer/promoters, although we did not test the other
C/EBP isoforms except for C/EBP.
is similar to that of human T-cell lymphotrophic virus type 1 tax
protein in its ability to stimulate the DNA binding of some bZip
proteins such as the ATF/CREB family protein and GCN4 (65, 66). The two
proteins also share the common feature of forming a complex with bZip
proteins only in solution, but dissociate under native gel
electrophoresis conditions (65). The human T-cell lymphotrophic virus
tax protein has been shown to recognize a basic region of the bZip
protein and to incorporate into the ternary complex as a dimer (66).
The detailed mechanism of HBx action should be studied further. HBx has
been shown to coactivate potent activation domains (9) and to interact
with basal transcription factors (13-16). In this respect, the
functional role of HBx in cooperation with C/EBP may include its
interaction with other activators or basal transcription machinery, not
merely enhancing the binding of C/EBP. This hypothesis may be
supported by the synergism between C/EBP and the acidic activation
domain of VP16 fused to the DNA-binding domain of HNF-1 (58) and also by the diverse interactions of C/EBPs with other transcription factors
to activate responsive promoters.
(Fig. 5B), but full-length HBx seems necessary for
functional cooperation with C/EBP (Fig. 5D). The
importance of the C-terminal activation domain of HBx in the
interaction with basal transcription factors (13-16) is relevant to
this result. The N-terminal regulatory domain of HBx was not necessary
for the direct interaction with C/EBP, and the C-terminal acidic activation domain of HBx alone was not sufficient. The importance of
the bZip domain of C/EBP in the interaction with HBx is consistent with the previous result on the interaction of HBx and CREB. It also
suggests a role of the bZip domain that is more than just dimerization
and DNA binding, which is also the case in the interaction of C/EBP
with other transcription factors.
ACKNOWLEDGEMENT
expression vector (pMSV-C/EBP).
FOOTNOTES
To whom correspondence should be addressed. Tel.: 82-2-880-6688;
Fax: 82-2-872-1993; E-mail: hyunerho{at}plaza.snu.ac.kr.
REFERENCES
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Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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