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(Received for publication, February 24, 1997, and in revised form, April 24, 1997)
From the Department of Molecular Biology and Research Center for
Cell Differentiation, Seoul National University,
Seoul 151-742, Korea
ABSTRACT The hepatitis B viral X promoter is known to be
positively autoregulated by its own HBx protein, which also interacts
with many cellular regulatory proteins. We investigated the effect of
activating transcription factor 2 (ATF2) on the activity of the X
promoter. Cotransfection of the ATF2 expression vector with a X
promoter-chloramphenicol acetyltransferase plasmid repressed the X
promoter activity in HepG2 cells. HBx activated activating protein 1 (AP-1)-mediated transcription through the hepatitis B virus E element
by 35-fold, while its activation activity was inhibited in the presence
of ATF2, suggesting that ATF2 inhibited the autoactivation of X
promoter by HBx and basal transcription mediated by AP-1. Since the
binding sites of AP-1 and ATF2 in the hepatitis B virus E element
overlap, the repression of X promoter activity by ATF2 is exerted by
the competition for the AP-1 binding site and the formation of the
ATF2-Jun heterodimer as in the case of the consensus AP-1 element.
However, the small X promoter had a ATF2 binding site and was activated
by ATF2. These results suggest that the syntheses of X proteins are
differentially regulated by ATF2.
INTRODUCTION Hepatitis B virus (HBV)1 is closely
related not only to acute and chronic hepatitis, but also to the
development of hepatocellular carcinoma (reviewed in Refs. 1 and 2).
HBV, unlike the other DNA viruses, encodes reverse transcriptase and
replicates through reverse transcription of pregenomic RNA (3, 4). The
production of pregenomic RNA is regulated by a combined interaction of
HBV enhancers (5, 6). One of the HBV genes, X, encodes a basic protein
of 154 amino acids. It has been implicated in the carcinogenicity of
this virus and is regarded as a major causative factor due to its
ability to induce hepatocellular carcinoma in transgenic mice (7). The
X protein exhibits a transcriptional activation function for many viral
and cellular genes without binding to DNA (8) (reviewed in Ref. 9). In
addition, a 3 One of the HBV enhancers, the EnI enhancer, partially overlaps with the
X gene promoter and is located between the S gene and the X gene coding
sequences (15, 16) (Fig. 1). This enhancer is composed of at least five
different factor binding sites named 2C, GB, EP, E, and NF1 sites
(17-21). A liver-enriched transcription factor, HNF3, binds to the 2C
site, and HNF4, RXR
We investigated the functional interaction between binding proteins on
the E element and the functional implication of X protein in the
regulation of the X promoter. Cotransfection of an ATF2 expression
vector with a X promoter-chloramphenicol acetyltransferase (CAT)
construct repressed the X promoter activity. Etkcat, a heterologous plasmid containing the E element in front of the tk promoter, was used
as a reporter for combinational cotransfection of ATF2, c-Jun, c-Fos,
and HBx expression vectors, which revealed that ATF2, by interfering
with AP-1, inhibits basal transcription of the X promoter and the
transactivation activity of HBx through AP-1. In addition, we also
tested whether the small X promoter activity is influenced by ATF2.
Although the X promoter was repressed by ATF2, the small X promoter had
a ATF2 binding site and was stimulated by ATF2. We conclude that
syntheses of X proteins are differentially regulated by ATF2.
EXPERIMENTAL PROCEDURES The X promoter-CAT reporter plasmid
(Xpcat) was constructed by insertion of the AccI (nt
1069)-BamHI (nt 1397) fragment of HBV-adr-k (25)
in front of the CAT gene. The mutant plasmid (XMpcat) has mutations in
the ATF2 binding site of E element which were generated by
oligonucleotide-directed in vitro mutagenesis (EM,
5 Proteins separated by SDS-polyacrylamide
gel electrophoresis were electrophoretically transferred to
nitrocellulose filters for 1 h at 20 °C using 5 V/cm2. Blocking was carried out in phosphate-buffered
saline (PBS) containing 5% defatted dry milk and 0.1% Tween 20, followed by washing and incubation of the membranes with antibodies in
the same buffer without defatted dry milk. Polyclonal X antibodies were
diluted 1:2,000 and used as primary antibodies. After incubation for
1 h at 25 °C, the blot was further incubated for 1 h at
25 °C with a horseradish peroxidase-linked anti-rabbit mouse
antibody. Protein/antibody complexes were visualized by the enhanced
chemiluminescence Western blotting detection system (Amersham Corp.)
according to the manufacturer's instructions.
Transient transfection
and CAT assay were performed as described previously (14).
Approximately 12 h prior to transfection HepG2 cells were plated
at a density of 1 × 106/60-mm diameter plate.
Transfection of plasmid DNA into HepG2 cells was carried out by the
calcium phosphate coprecipitation method as described previously (27).
Typically, 2 µg each of reporter and effector plasmids were used. The
total amount of transfected DNA was always adjusted to 12 µg with
pUC19. After 42 h, cells were harvested, and CAT assays were
performed. Cell extracts were normalized to the total amount of
protein, as determined by the Bradford assay (Bio-Rad). CAT enzymatic
activity was quantitated by measuring conversion of chloramphenicol to
its acetylated forms using the BAS radio analytic imaging system
according to manufacturer's instructions. All experiments were
repeated at least three times.
Escherichia coli cells were
grown in LB medium containing 50 µg/ml ampicillin. Subsequently the
cells were induced under vigorous shaking conditions with 0.5 mM isopropyl-1-thio- The pETc-jun DNase I footprinting analysis
was performed as described previously (14) with some modifications as
follow. Briefly, the SacII site (nt 1445) was cut and then
radiolabeled by incubation with [ RESULTS To study the effect of ATF2 on the activity
of the HBV X promoter, an expression plasmid encoding ATF2 cDNA was
cotransfected into HepG2 cells together with a X promoter-CAT construct
(Xpcat), or a construct having mutations in the ATF2 binding site of
the E element (XMpcat). As shown in Fig. 1A,
ATF2 repressed CAT expression from the X promoter two fold in HepG2
cells, but not with a construct having a mutation in the ATF2 binding
site. These data suggest that ATF2 repressed X promoter activity
through the ATF2 binding site. Since ATF2 is known to be an activator,
we investigated the effect of ATF2 on the consensus CRE cis-element in
a heterologous promoter system. The oligonucleotides of E and consensus
CRE were multimerized and inserted in front of the tk promoter. The
resulting plasmids were cotransfected with the ATF2 expression vector.
ATF2 activated CRE-mediated expression from the tk promoter 8-fold, whereas it repressed E element-mediated expression from the tk promoter
(Fig. 1B). It is well known that ATF2 activates CRE-mediated transcription (29). These results imply that other transcription factors may be the target for repression of the X promoter by ATF2.
The sequence of the E element (5
To assess this observation, we explored the physical interaction among
binding proteins on the E element with a mobility shift assay. The same
amounts of GST-Fos, GST-ATF2, c-Jun, and MBP-HBx were used for the
assays. Jun or Jun/Fos by themselves bound to the E element, although
binding efficiency was 5- to 7-fold lower (Fig. 2B,
lanes 1 and 2) than that of the consensus AP-1
element (Fig. 3A, lanes 1 and
2). But the addition of ATF2 protein resulted in the
diminished binding of Jun or Jun/Fos protein (Fig. 2B, lanes 4 and 5). In contrast, Jun alone decreased
the binding of ATF2 on the E element (Fig. 2B, lane
4), whereas Jun/Fos did not (Fig. 2B, lane
5). Addition of anti-Jun antibody to the mixtures of ATF2 and Jun,
or ATF2 and Jun/Fos resulted in the unchanged binding of ATF2 and no
supershifted complex (Fig. 2B, lanes 6 and
8). But the DNA-protein complexes were supershifted by
anti-ATF2 antibody (Fig. 2B, lanes 7 and
9). These data demonstrate that the binding of ATF2 to the E
element hindered the binding of AP-1 (Jun/Fos) probably due to the
overlapping binding sites in the E element. For the demonstration of
the ATF2's stronger affinity for the E element than that of AP-1, we
examined the effect of increasing amounts of ATF2 on the binding of
AP-1. When the amount of ATF2 increase from 20 ng to 100 ng to the
fixed 100 ng of AP-1 protein, less than 50 ng of ATF2 could completely
abolish the binding of AP-1 to the E element (Fig. 2C).
These results clearly showed that the ATF2 has a stronger affinity for
the E element than that of AP-1. MBP-HBx augmented the binding of ATF2
on the E element, but could not induce the binding of Jun or Jun/Fos in
the presence of ATF2 (Fig. 2B, lanes 10 and
11).
To examine the change of AP-1-mediated transcription by
ATF2, which does not bind well to the consensus AP-1 site, transient transfection and mobility shift assays were performed (Fig. 3). Jun and
Jun/Fos bound to the AP-1 site efficiently (Fig. 3A,
lanes 1 and 2), which was also confirmed by a
supershift by anti-Jun antibody (Fig. 3A, lane
5), but incubation of ATF2 with Jun prior to the addition of the
probe did not alter the mobility of the complex (Fig. 3A,
lanes 3 and 4) and no super-shifted band was detected with anti-ATF2 antibody (Fig. 3A, lane
6). This result suggests that ATF2/Jun heterodimer as well as ATF2
homodimer did not bind well to the consensus AP-1 site. However, the
addition of increasing amounts of ATF2 decreased the binding of Jun
homodimer on the AP-1 site (Fig. 3A, lanes
7-10). These results suggest that the formation of the ATF2/Jun
heterodimer decreased the formation of the Jun homodimer and
consequently diminished the binding of Jun homodimer on the AP-1 site.
We next examined the effect of ATF2 on the transcription mediated by
AP-1 binding sites (Fig. 3B). The decreased expression from
the tk promoter was observed by cotransfection of the ATF2 expression
vector with the APtkcat reporter, even though ATF2 alone could not bind
to the AP-1 site. Coexpression of Jun/Fos augmented the AP-1-mediated
transcription from tk promoter four-fold (Fig. 3B,
lane 3). The activation by Jun/Fos was further enhanced by
cotransfection with the HBx expression vector (Fig. 3B,
lane 4). However, coexpression of ATF2 diminished the
activation by Jun/Fos and the additional activation by HBx (Fig.
3B, lanes 5 and 6). In summary, ATF2
could also repress AP-1 activity in a manner consistent with a
protein-protein interaction.
It was also tested if the binding activity of ATF2 and c-Jun on the HBV
E element is affected by protein-protein interaction. To discriminate
the DNA-protein complexes of c-Jun, ATF2, and ATF2/c-Jun heterodimer,
the truncated c-Jun between amino acids 74 and 232 was obtained by
in vitro translation and used for the analysis. The
truncated c-Jun (c-Jun We investigated whether HBx affects the binding of Jun,
Jun/Fos and ATF2 on the E element (Fig. 2B, lanes
10 and 11). HBx enhanced the binding affinity of ATF2
to the E element 5-10-fold (Fig. 4A,
lanes 4 and 5). Smaller forms of X protein, which
have the C-terminal domain in common, were reported to activate class II and III promoters (13, 32). Smaller forms of X protein were also
examined for interaction with ATF2 (Fig. 4). Increasing amounts of
full-length X (Fig. 4A, lanes 4 and 5)
and middle X proteins (Fig. 4A, lanes 6 and
7) increased the binding of ATF2 on the E element. However,
small X protein (Fig. 4A, lanes 8 and 9) increased the ATF2-binding only slightly. To see whether
ATF2 and X proteins can interact with each other in the absence of a
cis-element, X proteins were incubated with glutathione-agarose beads
containing immobilized GST-ATF2, and then washed extensively with PBS
three times. The interacted proteins were eluted with PBS containing
0.1% triton X-100, and electrophoresed in a 8% SDS-polyacrylamide
gel. Bands corresponding to the full-length X and middle X proteins
were detected with anti-HBx antibody (Fig. 4B, lanes
2 and 3). However, only a faint band of small X protein was detected by the Western analysis (Fig. 4B, lane
4). These observations agree with that of the mobility shift assay
(Fig. 4A). Middle X and small X proteins have amino acid
residues 70-105 and 105-154 of full length X protein, respectively.
Therefore, amino acid residues 70-105 of X protein participates in the
interaction with ATF2. This domain of X protein corresponds to one of
three domains that are known to interact with cellular proteins (33). These data also indicate that X protein can interact with ATF2 in the
absence of a proper cis-element.
We next investigated
the effect of ATF2 on the activity of the small X promoter. A series of
small X promoter deletion constructs, described previously (14), was
cotransfected into HepG2 cells with the ATF2 expression vector. As
shown in Fig. 5A, expression from pHH115,
which has only a minimal promoter region, was still activated by ATF2
(Fig. 5A, lane 10), suggesting that the region between nt 1567 and 1679 is responsible for the activation by ATF2.
This region corresponds to the minimal small X promoter and was already
known to have several transcription factor binding sites, including
HNF4 and Sp1 (34, 35). To test whether this region has the binding site
for ATF2, a 115 base pair HpaII-HincII DNA
fragment was radiolabeled and used as a probe for a mobility shift
assay (Fig. 5B). Incubation of purified GST-ATF2 with DNA probe resulted in a specific DNA-ATF2 complex which competed with a
50-fold molar excess of E element, but not with the same amount of
mutant oligonucleotide, EM, indicating that ATF2 binds specifically to
the minimal small X promoter. To know the exact binding site of ATF2 on
the small X promoter, DNase I footprinting analysis was performed with
SacII-DraI DNA fragment (nt 1145-1719) as a probe. The protected region by ATF2 spanned from nt 1638 to 1656, and
have a sequence (TTACATAA) similar to the ATF2 binding site of
E-selectin promoter (TGACATCA) (36). These results suggest that this
protected region of the small X promoter is responsible for the
activation by ATF2.
DISCUSSION Two enhancers, EnI and EnII, have been identified in the HBV
genome (15, 16, 35, 37, 38). EnI is located in the region downstream of
the HBsAg ORF and upstream of the X ORF. Therefore, the transcription
from the X promoter was largely dependent on the EnI activity. EnI is
composed of multiple functional elements that act synergistically to
stimulate transcription. Two regions of E and EF-C in particular appear
to be important for EnI to exhibit a transacting function (17, 22, 39).
In this report, we analyzed the transcriptional regulation of X
promoter mediated by the E element of EnI. ATF2, one of the binding
proteins of the E element, repressed AP-1 and HBx-mediated
transcriptional activation of the X promoter. It was especially of
interest that EnI activity was negatively regulated by the
transcription factor ATF2, which is known to be a general activator.
Since AP-1 and ATF2 could bind to the same site of the E element
independently and were known to interact with each other, it is likely
that the down-regulation of the X promoter by ATF2 occurs via the
direct competition for factor binding sites and the formation of the ATF2/c-Jun heterodimer which can not bind efficiently to the E element.
Though some genes have an autonomous silencer element (40), others may
be repressed by the displacement of transactivator proteins from the
promoter element. In the case of the sea urchin histone H2B-1 gene, a
displacement protein appears to sterically prevent interaction of the
CAAT-binding protein (41). Direct protein-protein interaction between
members of different transcription factor families may be a general
mechanism by which a limited number of transcription factors can
specifically regulate a large number of genes. Gene repression by
protein-protein interaction was also observed in the HBV genes.
Addition of Jun, but not of Jun/Fos, in the incubation mixture reduced
the binding of ATF2 on the E element, indicating that the reduction of
ATF2 binding was caused not by the competition for binding sites but by
protein-protein interaction. A variant of the AP-1 binding site and of
CRE within the EnI of HBV could bind ATF2 and AP-1, respectively, but
the Jun/ATF2 heterodimer did not bind well (Fig. 3C,
lanes 8 and 9). The AP-1 binding sites within the
c-Jun (TTACCTCA) and ELAM promoters (TGACATCA) appear to specifically
bind a Jun/ATF2 heterodimer, whereas only AP-1 binds on the consensus
AP-1 binding site (TGAGTCA). Therefore, it appears that variants of
both the CRE and AP-1 consensus binding sites determine the binding
specificity of these sites for members of both the ATF/CREB and Jun/Fos
families.
The X gene product may stimulate its own synthesis through the E
element (42). If so, this would obviously require a mechanism to
down-regulate X expression after a desired level of X protein has been
reached. In agreement with such an autoregulatory scheme, a reduction
of HBV gene expression was observed if a high level of X gene product
was supplied in trans (43). A broad spectrum of X gene activity on
transcription suggests the possibility that its expression during viral
replication may have an influence on cellular genes that are involved
in the regulation of HBV transcription. We postulate that ATF2 is one
of the best candidates for the regulatory factor involved in the
auto-regulation of HBx production. Overexpression and activation of
ATF2 resulted in the enhanced binding of ATF2 and consequent repression
of the X promoter. ATF2 can be activated both by regulatory proteins
and through phosphorylation. Protein activators of ATF2 include
adenovirus E1a, HTLV-I Tax, and HMGI(Y) (44-47). The second ATF2
activation mechanism involves phosphorylation (48, 49). Phosphorylation
of ATF2 would lead to a conformational change in ATF2, resulting in an
increase of its DNA binding affinity. This possibility is consistent
with the observation that ATF2 binding to DNA can be correlated with
its phosphorylation state (50). In this context, it could be postulated
that HBx protein activates ATF2 both through direct interaction with
ATF2 and the phosphorylation of ATF2 by activating some kinases. These
observations suggest that activation of ATF2 by any pathway results in
the repression of X promoter, and the production of X protein can be
regulated automatically by ATF2.
Kwee et al. (13) found that smaller forms of X protein,
initiated at the second and the third ATG codons of the X ORF, also have the transactivating function. Small X promoter and its positive regulatory element was reported to be located in the 5 FOOTNOTES REFERENCES
Volume 272, Number 27,
Issue of July 4, 1997
pp. 16934-16939
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-truncated X gene-cell fusion product from integrated HBV
DNA in chronic hepatitis tissue (10) and a 3-elongated X product of
193 amino acid (21 kDa) from mutant HBV DNA (11, 12) were shown to
exert a transactivation function. Smaller forms of the X protein,
initiated at the second and the third ATG codons of the X ORF, are also important in the transacting function. All three forms of the X protein
can individually transactivate a class III promoter, which transcribes
the tRNA and 5 S rRNA by RNA polymerase III. In contrast,
transactivation of several different class II promoters, which
transcribe the mRNAs by RNA polymerase II, displays various requirements for the different X proteins (13). The small X promoter
expresses the smaller X gene transcripts that arise within the X ORF
and whose heterogeneous 5 ends straddle the second ATG codon. A
positive regulatory element is also known to be necessary for the
efficient transcription from the small X promoter (14).
and COUP-TF bind to the GB element in
vitro (20). Nuclear factor EF-C and NF1 bind to the inverted
repeat of the EP element and NF1 site, respectively (22). The E element
is bound by ATF2/CREB and AP-1, and also mediates the transactivation
of X promoter activity by X protein. Deletion, linker scanning, and
point mutation analyses of the E element revealed that mutation of the
E element reduced X promoter activity by 20-50% (23, 24).
Fig. 1.
The effects of ATF2 on the hepatitis B viral
X promoter. A, schematic diagram of EnI and X promoter is
shown at the top, in which representative cis-elements of
2C, GR, EP, E, and NF1 are shown. The histogram represents the relative
levels of CAT activity in extracts of HepG2 cells cotransfected with 2 µg each of reporter plasmids and 5 µg of ATF2 expression plasmid as
indicated. B, CAT assay using the heterologous reporter
plasmids. The ATF2 expression plasmid was cotransfected with
heterologous reporter plasmids, Etkcat and CREtkcat, which have five
copies of E oligonucleotide and three copies of consensus CRE
oligonucleotide, respectively, in front of tk promoter. Two micrograms
and one microgram of Etkcat and CREtkcat, respectively, were
cotransfected into HepG2 cells with 5 µg of ATF2 expression vector as
indicated.
[View Larger Version of this Image (22K GIF file)]
-CTGCCAAGTATTTGCTAATTCAACCCCCA-3). The
mutated sequences are underlined. The Etkcat was constructed by
inserting pentamerized double stranded E
(5-CTGCCAAGTATTTGCTGACGCAACCCCCA-3) oligonucleotide into the
SmaI site of pBLcat2. APtkcat and CREtkcat were constructed by inserting trimerized double stranded consensus AP-1
(5-CGCTTGATGAGTCAGCCGGAA-3) and CRE
(5-AGAGATTGCCTGACGTCAGAGAGCTAG-3) oligonucleotides, purchased from
Promega (Madison, WI), into the SmaI site of pBLcat2, respectively. Eukaryotic expression plasmids of HBx, the small X
promoter-CAT construct and serial deletion constructs were described previously (14). The ATF2 eukaryotic expression vector was constructed by subcloning the full-length ATF2 cDNA into the pECE expression vector (26). The c-Jun and c-Fos expression vectors were kindly provided by K. Kim (Seoul National University, Korea). Bacterial expression vectors for ATF2 and c-Fos were constructed by inserting their cDNAs in frame into pGEX-3X. MBP-fused X, middle X, and small
X expression vectors were obtained by inserting full-length and deleted
fragments of X genes into the pMAL-c2 plasmid, resulting in proteins
which have amino acid residues 1-154, 70-154, and 105-154 of X
protein, respectively. For the construction of expression plasmid of
truncated c-Jun (pETc-jun
), the c-jun gene was subcloned into the vector pET3b (Stratagene) to generate the plasmid pETc-jun and
subsequently deleted the DNA fragment between the two AvaI sites (nucleotides 220 and 679 of the c-jun gene), which
resulted in the deletion of amino acids between 74 and 232 of c-Jun
protein.
-D-galactopyranoside for
3 h at 30 °C. GST fusion proteins were purified according to
the protocol from Pharmacia Biotech Inc., and MBP-fused X proteins were
purified according to the Short Protocols in Molecular
Biology (28). DNA binding reactions were carried out in a 15-µl
volume, which typically contains 10 mM Hepes (pH 7.9), 60 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol,
500 ng of poly(dI-dC) (Sigma), and 2 µg of bovine serum albumin.
Prior to the reaction with DNA, 100 ng each of c-Jun, GST-Fos,
GST-ATF2, and MBP-HBx proteins were mixed and incubated for 20 min.
Antibodies against ATF2 and c-Jun were from Santa Cruz Biotech. Inc.
(Santa Cruz, CA). DNA binding was started by adding 10,000 cpm of probe to the preincubated reaction mixture with incubation for 15 min at room
temperature. Samples were loaded on a 4% polyacrylamide gel
(acrylamide:bisacrylamide = 60:1) in 0.5 × TBE (44 mM Tris, 44 mM boric acid and 1 mM
EDTA). After electrophoresis, gels were dried and exposed to x-ray
film.
plasmid was linearized and used for the coupled in vitro
transcription and translation reaction. Truncated c-Jun protein was
synthesized in rabbit reticulocyte lysates programmed with the in
vitro transcribed mRNA in the presence of unlabeled methionine
according to the protocol recommended by the manufacturer (Promega).
-32P]ATP (specific
activity, 7000 Ci/mmol) after dephosphorylation with calf intestinal
phosphatase. The radiolabeled DNA fragment was cut with DraI
(nt 1719), fractionated on an 6% polyacrylamide gel and isolated by
electroelution for DNase I footprinting.
-TGACGCAA-3)
is almost identical to consensus CRE and AP-1 binding sites. Also, the
bindings of AP-1 and ATF2 on this element were reported previously (30, 31). To see the repression mechanism of ATF2, we cotransfected c-Jun,
c-Fos, ATF2, and HBx expression vectors in various combinations into
HepG2 cells with Etkcat as a reporter plasmid, and asked whether ATF2
can inhibit AP-1 activity and HBx-mediated transcriptional activation.
The Jun homodimer and Jun/Fos heterodimer activated the reporter
plasmid about 9- and 7-fold, respectively (Fig.
2A, lanes 3 and 5,
respectively). The activation property was increased with coexpression
of HBx 25- and 35-fold, respectively (Fig. 2A, lanes
11 and 13, respectively). But the expression of Fos
alone exhibited no significant activation with or without the
expression of HBx protein (Fig. 2A, lanes 12 and
4). Cotransfection of ATF2 expression vector repressed the
transactivation by Jun and Jun/Fos (Fig. 2A, compare
lanes 3 and 5 with lanes 7 and
9, respectively). This repression of Jun and Jun/Fos by ATF2
was also shown in the additional activation by HBx (Fig. 2A,
compare lanes 11 and 13 with lanes 14 and 16, respectively). It is of interest that ATF2 repressed
the activity of the Jun/Fos heterodimer (Fig. 2A,
lanes 9 and 16) more than that of the Jun
homodimer (Fig. 2A, lanes 7 and 14)
especially with additional activation by HBx.
Fig. 2.
ATF2 represses the activation activity of
AP-1 and HBx-mediated activation. A, transcriptional
activities of Etkcat were measured in HepG2 cells. Two micrograms of
Etkcat reporter plasmid were cotransfected into HepG2 cells with 2 µg
each of plasmids expressing c-Jun, c-Fos, ATF2, and HBx proteins as
indicated. Cell extracts from transfected cells were normalized to the
total amount of protein as determined by Bradford assay (Bio-Rad). CAT assays were repeated four times and a representative CAT assay is
shown. B, binding assay with c-Jun, c-Fos, and ATF2 on the HBV E element. Bacterially expressed and purified c-Jun, GST-c-Fos, GST-ATF2, and MBP-HBx were incubated in the presence of 2 µg of bovine serum albumin and 500 ng of poly(dI-dC) for 15 min at room temperature as indicated, followed by adding a probe. The same amount
(100 ng) of each protein was used in the assay. For antibody supershift
analysis, 1 µl of each antibody was added and further incubated for
15 min at room temperature prior to electrophoresis. C,
competition assay between ATF2 and AP-1 for the binding on HBV E
element. Mobility shift assay was accomplished for the E element as a
probe with increasing amounts of ATF2 (0, 20, 50, and 100 ng) to the
fixed 100 ng of AP-1 (50 ng each of c-Jun and GST-Fos).
[View Larger Version of this Image (57K GIF file)]
Fig. 3.
The effects of ATF2 on the consensus AP-1
binding site. A, binding of Jun/Fos on the consensus AP-1
binding site in the presence of ATF2. Bacterially expressed GST-ATF2,
c-Jun, and GST-Fos were incubated with 32P-labeled AP-1
oligonucleotide as a probe. The amounts of each protein used were 100 ng for lanes 1-6. In lanes 7-10, the amounts of
ATF2 used were 0, 50, 100, and 150 ng, respectively, to the fixed 50 ng
of c-Jun. The detailed procedure of mobility shift assay and antibody
supershift assay were the same as described in the legend of Fig. 2.
B, the effect of ATF2 on the AP-1-mediated tk promoter
activity. One microgram of APtkcat reporter plasmid was cotransfected
into HepG2 cells with 2 µg of each plasmid expressing c-Jun, c-Fos,
ATF2, and HBx as indicated. C, mobility shift assay using
ATF2/c-Jun heterodimer with CRE and HBV E element as probes. Five
microliter of in vitro translated c-Jun, 100 ng of ATF2 protein (lanes 2, 3, 7, and
8) and 200 ng of ATF2 (lanes 4, 5, 9, and 10) were used. The mixture of ATF2 and
c-Jun was incubated for 20 min at 30 °C for the efficient
formation of heterodimer, followed by adding a probe. For supershift
analysis, anti-ATF2 antibodies were added after addition of the probe
(lanes 5 and 10).
[View Larger Version of this Image (41K GIF file)]
) contains the intact DNA-binding domain and
leucine zipper domain which is responsible for dimerization with other
proteins. The ATF2/c-Jun heterodimer, which migrates between
c-Jun and ATF2, bound well on the CRE element (Fig. 3C, lanes 3 and 4) but did not bind efficiently on
the HBV E element (Fig. 3C, lanes 8 and
9). Therefore, ATF2 could repress transcription mediated by
cis-elements such as consensus AP-1 site and HBV E element on which
ATF2/c-Jun heterodimer do not bind efficiently.
Fig. 4.
Physical interaction of HBx with ATF2.
A, a constant amount (100 ng) of ATF2 protein was incubated with
each of increasing amounts (1 µg, lanes 4, 6,
and 8; 2 µg, lanes 5, 7, and
9) of MBP-HBx, MBP-Mx, and MBP-Sx proteins in the presence
of 500 ng of poly(dI-dC) and 2 µg of bovine serum albumin, prior to
addition of 32P-labeled E oligonucleotide as a probe. For
competition analysis, 100-fold molar excess of E (lane 2)
and mutated oligonucleotide, EM (lane 3) were used.
B, each of MBP-HBx, MBP-Mx, MBP-Sx, and MBP proteins was
incubated with glutathione-agarose beads containing immobilized
GST-ATF2, and then washed extensively with PBS three times. The
interacted proteins were eluted with PBS containing 0.1% Triton X-100,
and electrophoresed in a 8% SDS-polyacrylamide gel. After
electrotransfer of proteins to a nitrocellulose membrane, the proteins
were detected by anti-X antibody as described under "Experimental
Procedures."
[View Larger Version of this Image (33K GIF file)]
Fig. 5.
The effect of ATF2 on the small X promoter.
A, a series of deletion constructs containing the regulatory
region of small X promoter was cotransfected into HepG2 cells with the
ATF2 expression vector as indicated. For the relatively low promoter activity, 2 µg each of pBH284 and pXH245 plasmids, and 5 µg each of
pXH215, pXH172, and pHH115 reporter plasmids were cotransfected into
HepG2 cells with 10 µg of ATF2 expression vector. CAT assay was
performed as described in Experimental Procedures. B,
binding of ATF2 on the HpaII-HincII (nt
1567-1679) DNA fragment. The HpaII-HincII DNA
fragment, which corresponds to the promoter region of pHH115 reporter
plasmid, was excised, eluted and radiolabeled with
[-32P]ATP after dephosphorylation with calf intestinal
alkaline phosphatase. The GST-ATF2 was incubated with the radiolabeled
HpaII-HincII DNA fragment as a probe. For
competition analysis, 50 ng each of E (lane 2) and EM
(lane 3) oligonucleotides was added in the reaction mixture.
C, DNase I footprinting analysis of small X promoter region
with ATF2. The SacII site (nt 1445) was cut and then
radiolabeled by incubation with [-32P]ATP after
dephosphorylation with calf intestinal phosphatase. The radiolabeled
DNA was excised with DraI (nt 1719), eluted and used as a
probe. Serial increasing amounts of ATF2 (0.5 and 60 µg) was used for
analysis as indicated. Protected region by ATF2 is denoted as a
solid bar.
[View Larger Version of this Image (38K GIF file)]
-distal half of
the X ORF (14). ATF2 activated the small X promoter through the region
responsible for the minimal small X promoter activity, while repressing
the X promoter through the E element of EnI, suggesting that
differential regulation of X proteins was exerted by ATF2. In our
previous report (14), we suggested that the X promoter may be regulated
by downstream DNA sequences corresponding to the small X promoter.
Although all three forms of the X protein can individually
transactivate the class III promoter, transactivation of several
different class II promoters displays various requirements for the
different X proteins. Therefore, it is conceivable that the X protein
and smaller forms of X protein may have different regulatory
activities. In summary, differential regulation of X proteins by ATF2
would result in the expression of different forms of X protein and a
different set of class II promoter would be modulated by X
proteins.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Seoul National University, Seoul 151-742, Korea. Tel.: 82-2-880-6688; Fax: 82-2-872-1993.
1
The abbreviations used are: HBV, hepatitis B
virus; ORF, open reading frame; AP-1, activating protein 1; ATF2,
activating transcription factor 2; CRE, cAMP-response element; CREB,
cAMP-response element binding protein; EnI, hepatitis B virus enhancer
I; GST, glutathione S-transferase; MBP, maltose-binding
protein; PBS, phosphate-buffered saline; nt, nucleotide; CAT,
chloramphenicol acetyltransferase; tk, thymidine kinase.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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