(Received for publication, March 12, 1997, and in revised form, May 21, 1997)
From the Department of Basic Medical Sciences, Purdue
University, West Lafayette, Indiana 47907 and the
§ Ohio State Biochemistry Program and Department of Medical
Biochemistry and Neurobiotechnology Center, Ohio State University,
Columbus, Ohio 43210
The hepatitis B virus X protein interacts with
the basic-region, leucine zipper protein (bZip) domain of cAMP response
element-binding protein increasing its affinity for the cAMP response
element site in vitro and its transcriptional efficacy
in vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819-3823).
Here we examine pX interactions with bZip transcription factors
ATF3, gadd153/Chop10, ICER II, and NF-IL6. We
demonstrate direct interactions in vitro between pX and the
bZip proteins tested. In contrast MyoD and Gal41-147 fail
to interact with pX. We also demonstrate by the mammalian two-hybrid
assay the direct interaction of pX with cAMP response element- binding
protein, ICER II
, ATF3, and NF-IL6 in hepatocytes. In
addition, pX increases the DNA binding potential of bZip proteins for
their cognate DNA-binding site in vitro. In transient
transfections in hepatocytes (AML12 cell line), pX increases the
transcriptional efficacy of the bZip transcription factors.
NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX.
Most interestingly, pX augments the repression mediated by bZip
repressors ATF3 and ICER II
, by 6- and 7-fold,
respectively, demonstrating for the first time the involvement of pX in
gene repression. We conclude that pX is an enhancer of the DNA binding
potential of bZip transcription factors, thereby increasing the
transactivation or repression efficacy of bZip-responsive genes.
The hepatitis B virus genome encodes a 16.5-kDa protein, termed X antigen (2), expressed during viral infection (3-5) and required for the viral life cycle (6, 7). Expression of pX in liver of transgenic animals has been shown in some cases to induce liver cancer (8), implicating pX in the development of hepatocarcinogenesis in humans chronically infected with HBV.1 However, the mechanism of hepatocarcinogenesis remains unknown.
Many studies have examined the role of pX in cellular signaling, but
its mechanism of action remains obscure. HBV X is a multifunctional protein; it interacts in vitro and in vivo with
the tumor suppressor p53 protein (9-11), with the putative DNA repair
protein XAP-1 (12), and acts as a promiscuous transactivator (13). pX
transactivates cis-acting elements, such as those present within the
HBV (14, 15) and SV40 enhancers (16, 17), and the HIV-LTR (18-21). X-responsive elements include the NF-B (18, 20, 21-23), AP-1 (24-28), AP-2 (24), and CRE (1, 29) sites. Interestingly, pX does not
appear to bind double-stranded DNA. These multiple X-responsive,
cis-acting elements suggest that the mechanism of pX action is
pleiotropic. Studies to date support pX transactivation by a dual
mechanism, i.e. both the activation of cytoplasmic signaling pathways and direct interactions with cellular transcription factors and the transcriptional machinery. Regarding this dual mechanism, pX
activates in vivo the Ras, Raf, mitogen-activated protein
kinase, and JNK signaling pathways, leading to transactivation of AP-1 and NF-
B sites (27, 30, 31).
pX interacts with several members of the basal transcriptional apparatus, such as the RPB5 subunit of eukaryotic RNA polymerases (32), TBP (33), and with components of TFIIH (34). Furthermore, it has been proposed that pX is a general viral transactivator, acting by influencing the coactivator process (35).
Direct interactions between pX and cellular transcription factors have been uniquely demonstrated for the CREB/ATF family of proteins (1, 29). CREB, which mediates the transcriptional induction of the cAMP-transduction pathway (36, 37), is also the interaction target of the HTLVI Tax oncoprotein (38, 39). Our studies demonstrated that the interaction of pX with CREB increases by 1 order of magnitude its affinity for the CRE site, thereby increasing its in vivo transcriptional efficacy by 13-fold. In contrast to the CREB/Tax interactions (40), pX does not enhance the dimerization rate of CREB; it seems to target the basic, DNA-binding region of CREB (1).
In this study we investigate the potential interaction of pX with other
bZip transcription factors normally expressed in the hepatocyte. Our
hypothesis is that pX interacts not only with CREB but also with other
bZip proteins, by recognizing common structural features shared by bZip
proteins (41). To test this hypothesis, we selected for analysis bZip
proteins that play a role in hepatocyte physiology such as, NF-IL6 or
C/EBP (42), gadd153/Chop10 (43), ATF3 (44), and ICER
II
(45, 80) which may play such a role.
NF-IL6, a member of the C/EBP leucine zipper family (42), is selected in this study, since it is expressed in liver (46) where it is implicated as the master regulator of the acute phase response genes (42). Expression of NF-IL6 is induced by IL-6 (42, 47), IL-1 (48), and other inflammatory mediators. NF-IL6 forms heterodimers with other bZip proteins in the liver (43), including gadd153/Chop10 (43) which is induced by cellular stress conditions (49, 50), acute phase (inflammatory) response (51), and exposure to toxins (52, 53). While gadd153/Chop10 has strong sequence similarity to C/EBP-like proteins within the bZip region (43), it contains substitutions of three conserved residues in the basic region critical for DNA binding. Therefore, heterodimers of gadd153/Chop10 and C/EBP-like proteins are unable to bind to their cognate DNA binding site (43). Accordingly, it has been proposed that gadd153/Chop10 functions as a stress-inducible transcriptional inhibitor (43). However, recent studies indicate that in certain cases gadd153/Chop10 may function as a direct transcriptional activator (54).
ATF3, is also of interest in this study since it is a member of the CREB/ATF family of transcription factors, sharing sequence similarity with the bZip domain of CREB and binding to the CRE site (44). However, while CREB is a positive regulator of transcription, ATF3 is a transcriptional repressor (56). Recent studies indicate that ATF3 is induced by a variety of physiological stress conditions, such as mechanically injured and toxin-injured liver (55, 57). ATF3 and gadd153/Chop10 form non-DNA-binding heterodimers (55). Interestingly, during carbon tetrachloride injury of liver, gadd153/Chop10 and ATF3 mRNAs are inversely induced but in an overlapping manner; gadd153/Chop10 mRNA, high in normal liver, decreases upon CCl4 exposure; ATF3 mRNA, low in normal liver, increases upon CCl4 exposure (55).
ICER II (inducible cAMP early
repressor) (45), a member of the cAMP-responsive element
modulator subfamily of bZip proteins, is of interest since it is an
inducible repressor, displaying high degree of amino acid sequence
identity with the bZip of CREB and is devoid of a transactivation
domain. Thus, ICER II
provides an ideal model system to demonstrate
the effect of pX in repression of bZip transcription. Recent studies
demonstrated ICER II
expression in liver regeneration (80).
In this study, we employ in vitro and in vivo (cellular) assays to examine the interaction of pX with the aforementioned bZip transcription factors.
Transcription
factors ICER II (45), the bZip domain of NF-IL6, amino acid residues
259-335, and CREB327 (59) were cloned in T7-7
vector. ATF3 (55) and gadd153/Chop10 (55) were in a
derivative of pTM1 (60) vector. 35S-Labeled proteins were
synthesized by the TNT (Promega) in vitro transcription/translation system. Plasmids GST-X1-154 and
GST-X49-154 were constructed by polymerase chain reaction
of pX DNA fragments corresponding either to amino acid residues 1-154
or 49-154 cloned into the EcoRI-HindIII sites of
plasmid pGEX-KG (61). GST-X fusion proteins were expressed in
Escherichia coli and purified on glutathione-Sepharose 4B
resin (Pharmacia Biotech Inc.), as described previously (62). Bacterial
extract obtained from 1 liter of bacterial culture was bound to 500 µl of resin for 30 min and washed (62). Protein concentration of GST
and GST-X1-154 bound to the resin was estimated by
Coomassie Blue staining of SDS-PAGE and by comparison to known amounts
of bovine serum albumin run on the same gel. In vitro
protein-protein interaction assays were carried out as follows: 10 µg
of GST and 2 µg of GST-X1-154 proteins immobilized onto
20 µl of glutathione-Sepharose 4B resin were incubated with 4 µl of
TNT lysate for 3 h at 4 °C, in buffer containing 25 mM Hepes, pH 7.5, 100 mM KCl, 5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 40 µg/ml bovine serum albumin, 0.1% Triton X-100, and a protease
inhibitor mixture. After incubation, the beads were washed six times in
the above buffer. Analysis of the bound protein was by SDS-PAGE and
fluorography.
DNA protein binding
assays were carried out as described previously (1). The somatostatin
CRE (62, 63) was used as the oligonucleotide probe for CREB, ICER
II, ATF3, and gadd153/Chop10. The nucleotide sequence
spanning positions
165 to
173 of the HIV-LTR (58) was used as the
DNA-binding probe for NF-IL6. The MyoD/E47 E-box
DNA-binding site (64) was kindly provided by Dr. S. Konieczny. Proteins
for ICER II
, ATF3, gadd153/Chop10,
ATF3/gadd153/Chop10, and MyoD/E47 were obtained
by in vitro translation (TNT, Promega); 1-3
µl of in vitro translation mixture was used for each
reaction. Bacterially produced CREB327 (70) and bZip
NF-IL6, 15 ng each, were employed in the binding reactions. Recombinant
GST-X49-154 was added to binding reactions; control lanes
contained equal amounts of GST protein. The reaction mixtures were
analyzed by native acrylamide gel electrophoresis, as described (62,
63).
AML12 cells were kindly provided by Dr. N. Fausto. AML12 cells were propagated in Dulbecco's modified Eagle's medium/Ham's F-12, supplemented with 10% fetal calf serum, a mixture of insulin, transferrin, and selenium (Life Technologies, Inc.), 0.1 µM dexamethasone, and gentamicin, 50 µg/ml (65).
In functional assays, AML12 cells were transfected by the calcium
phosphate coprecipitation method, using the Life Technologies, Inc.
transfection kit. Briefly, 10-20% subconfluent cultures were transfected with 5 µg of CAT reporter plasmid and the indicated amount of expressor plasmid. Cells were harvested 48 h after
transfection. CAT activity was determined as described (66) using equal
amounts of protein extract per assay. Protein concentration of cellular extracts was determined by the Bio-Rad protein assay. Each experiment was repeated a minimum of three times. The CAT reporter vector pC15XE, kindly provided by Dr. M. Bina, contains only the
NF-IL6-binding site I, at position
158 to
178 (58).
The mammalian two-hybrid assay was carried out using the 5 × Gal4-E1b TATAA-luciferase reporter plasmid (67). The
RSV-CREB-VP16 and RSV-Gal4 vectors were kindly provided by Dr. R. Gaynor (68). The CMV4-ICER II (45) was kindly provided by Dr. C. Molina. The ATF3, gadd153/Chop10, ICER II
, and bZip of
NF-IL6 (amino acid residues 259-335) were cloned by polymerase chain
reaction amplification of the respective fragments into the RSV-VP16
vector (68), at the NcoI site, resulting in the construction
of bZip-VP16 fusion proteins, as described by Yin et al.
(68). Similarly, RSV-X-Gal4 constructs were prepared by inserting the
coding region of pX at the NcoI site, resulting in fusion
proteins with the Gal4 DNA-binding domain (amino acids 1-147) at its C
terminus. 5 µg of reporter plasmid, 5 × Gal4-E1b
TATAA-luciferase, were cotransfected with 5-10 µg of each of the
expressor plasmids. Transfections were performed in duplicates (60-mm
plates) and repeated a minimum of three times.
Interactions between pX and CREB were demonstrated by enhanced CREB binding to the CRE site in the presence of pX (1, 29) and by altered methylation interference assays (1). In the present study we employed the CREB/pX-interacting proteins as the model system for developing an assay to detect direct protein-protein interactions. For this analysis the full-length X protein was produced as a fusion with glutathione S-transferase (61). The GST-X1-154 fusion protein is selectively retained by glutathione-Sepharose 4B resin and thus the complex provides a suitable affinity resin.
Recombinant CREB327 (69, 70), 32P-radiolabeled
(66), was employed to establish the in vitro conditions for
detecting specific binding of CREB327 to the
resin-immobilized GST-X1-154. We observe selective binding
of 32P-CREB327 to GST-X1-154 but
no binding to the control GST-resin (Fig.
1A). Additional control
experiments include the following: first, the demonstration that under
the conditions detecting CREB/pX interactions (Fig. 1A),
transcription factors of a different class, namely
Gal41-147 (71) and MyoD (64), did not display detectable
binding to GST-X1-154 (Fig. 1, B and
C); and second, the demonstration that the interaction of
CREB327 with GST-X1-154 involved the bZip
domain of CREB327. For this analysis, CREB327
and the N-terminal portion of CREB327, amino acid residues
1-198, were synthesized in vitro (Fig.
2A). In comparative analysis,
the N-terminal region of CREB327 did not interact with
GST-X1-154 (Fig. 2B), whereas
35S-CREB327 displayed specific binding to
GST-X1-154 (Fig. 2C). The results confirm our
earlier observations that the bZip of CREB is the interacting target of
pX and show that under the established conditions (Fig. 1) we detect
specific binding of CREB327 to pX in vitro.
We employed the in vitro protein-protein interaction assay
described in Fig. 1A to examine the interaction of bZip
transcription factors ATF3, gadd153/Chop10, NF-IL6, and
ICER II with pX. The binding reactions with bZip proteins were
carried out exactly as described for CREB327 (Fig.
1A and Fig. 2C). Fig.
3A shows the binding of the
bZip of NF-IL6 to the immobilized GST-X1-154 is much
higher than the control GST-resin. Similarly, Fig. 3, C and
D, shows the specific binding of ATF3,
gadd153/Chop10, and ICER II
to GST-X1-154. These
observations demonstrate that pX can interact directly with bZip
transcription factors.
The Effect of HBV pX on the DNA Binding Potential of bZip Proteins
Gel retardation assays demonstrated that CREB/pX interactions increase the DNA binding affinity of CREB for the CRE site (1). Therefore, we carried out DNA-binding assays to examine the effect of pX on the DNA-binding activity of the bZip proteins (Fig. 3). We incubated the bZip proteins with their cognate radiolabeled DNA-binding site, as a function of recombinant pX addition. The pX used is a truncated form of the full-length protein, lacking the N-terminal 48-amino acid residues. We constructed this truncation based on observations by Murakami et al. (72) that the N-terminal deleted portion does not affect pX transactivation. In the protein-protein interaction assay GST-X49-154 behaves similarly to X1-154 and interacts equally with all the bZip proteins factor tested.2 Furthermore, similar to pX1-154, pX49-154 enhances equally the DNA binding activity of CREB (data not shown). We employed the GST-X49-154 because, in our hands, it displays consistent activity and does not precipitate as readily as pX1-154 or GST-X1-154.
Fig. 4 shows enhanced DNA binding
in the presence of pX49-154 for CREB, in agreement
with our earlier results (1), and for ICER II, ATF3, and
NF-IL6. Interestingly, neither the non-DNA-binding gadd153/Chop10 nor
the ATF3/gadd153/Chop10 heterodimer displays enhanced DNA
binding by pX. Likewise, the MyoD/E47 heterodimer, used
here as the negative control, displays only minimal increase in DNA
binding in the presence of pX, demonstrating the preference of pX for
interaction with bZip transcription factors.
Furthermore, our observations suggest that the bZip transcription
factors, which are capable of both DNA binding and protein-protein interactions with pX (CREB, ICER II, ATF3, and NF-IL6)
(Figs. 3 and 4), display enhanced DNA binding in vitro in
the presence of pX. However, pX does not convert a non-DNA-binding
protein, such as gadd153/Chop10, to become DNA binding. pX only
potentiates the existing DNA binding activity of bZip proteins.
Similarly, the non-bZip transcription factors GAL41-147
(1) and MyoD/E47, which fail to directly interact with pX
(Fig. 3), do not acquire enhanced DNA binding by pX. We conclude that
pX interacts directly and specifically with bZip transcription factors
and increases their DNA binding potential.
To further establish direct interactions between pX and bZip
transcription factors in the hepatocyte, we employed the mammalian two-hybrid assay, in the AML12 cell line (65). AML12 cells, derived
from transgenic mouse liver, over-express transforming growth
factor-, but otherwise exhibit properties of normal hepatocytes (65). For this analysis we constructed an expression vector containing the activation domain of VP16412-490 in
fusion with the C terminus of CREB (68), ICER II
, ATF3, and NF-IL6. We also constructed a mammalian expression vector encoding
pX in fusion at its C terminus to the DNA-binding domain of
Gal41-147. Transient transfections of RSV-X-Gal4 in AML12
cells demonstrated that the pX-Gal4 fusion protein is devoid of
detectable transactivation potential (Fig.
5).
Fig. 5 shows the relative amount of reporter expression following
cotransfection of both RSV-X-Gal4 and RSV-bZip-VP16 expressors, when
compared with control transfections. With cotransfection of either
RSV-CREB-VP16 or RSV-ICER II-VP16 with RSV-X-Gal4 plasmid, we
observe a 3- and 6-fold induction of luciferase expression, respectively. Cotransfection of RSV-NF-IL6-VP16 or
RSV-ATF3-VP16 with RSV-X-Gal4 induces the
expression of luciferase by 4- and 14-fold, respectively. To
verify the specificity of our in vivo assay, we
employed the pEM-MyoD-VP16 expressor as our control, kindly
provided by Dr. S. Konieczny.3 We observe no
detectable interactions occurring in vivo between MyoD and pX by this assay. These observations agree with our
in vitro results with MyoD (Figs. 1 and 4). We
conclude that in vivo pX interacts directly via
protein-protein interactions with the bZip proteins CREB, ICER
II
, ATF3, and NF-IL6 (Fig. 5).
Having demonstrated that pX interacts directly
with bZip proteins in the hepatocyte (Fig. 5), we performed
transient transfection assays to assess the effect of these
interactions on the transcriptional activity of the
aforementioned bZip proteins. Among the bZip proteins, NF-IL6
is a well documented transcriptional activator (73). Earlier studies by
Tesmer et al. (58) identified the cis-acting element located
between nucleotide position 158 to
178 on the LTR of HIV as an
NF-IL6 binding site. We employed an HIV-LTR-driven reporter, pC15
XE
(58), as the model system for analyzing NF-IL6-mediated transcriptional
induction. A representative experiment is shown in Fig.
6A. AML12 cells were
transfected under low serum conditions in the presence of 10 nM IL-6 (73). Increasing amounts of CMV4-X expressor were
transfected in the presence or absence of CMV4-NF-IL6 vector (58). We
observe a 3-fold enhancement in HIV-LTR-CAT transcription by pX only in
the presence of the NF-IL6 encoding vector, suggesting that pX action
augments the transcriptional efficacy of NF-IL6. In contrast, the pX
encoding vector failed to demonstrate enhanced transcriptional
induction of a MyoD-dependent transcription system (74),
indicating that pX enhances transactivation in a specific manner (Fig.
6B).
HBV pX Enhances the Transrepression Potential of bZip Repressors
We have likewise employed transient transfections in
AML12 cells to examine the effect of pX on the bZip repressors ICER
II and ATF3. In our in vitro assays (Figs. 3
and 4) we included the bZip inhibitor gadd153/Chop10. Although pX
interacted with gadd153/Chop10 in vitro (Fig. 3), we did not
further analyze gadd153/Chop10 because pX did not alter or augment its
DNA binding potential (Fig. 4).
The assay system used to monitor the effect of pX on bZip repressor
activity includes the CRE3-CAT reporter plasmid and the
ATF3 (45) and ICER II (55) expressor plasmids. The pX
expression vector encodes the nuclear localization sequence (NLS) in fusion with pX
(30) which targets pX exclusively in the nucleus. Titration experiments
of increasing amounts of bZip repressor were carried out in the
presence or absence of CMV-NLS-pX plasmid which was used
interchangeably with CMV4-X, as in Fig. 6.
In Fig. 7, CRE3-CAT reporter
activity was monitored in the presence of forskolin which activates the
endogenous CREB (1, 66). Transfection of increasing amounts of ICER
II expressor in the absence of pX expression results in a 2-8-fold
inhibition of CRE-driven transcription. In agreement with our earlier
observations (1), transfection of pX, in the presence of forskolin,
induces the transcriptional efficacy of endogenous CREB, from 13.5 to 81%. In Fig. 7, we show that increasing amounts of transfected ICER
II
DNA in the presence of pX expression resulted in 7-23-fold repression. Therefore, the repression is higher in the presence of pX
(7-23-fold) than that in the absence (2-8-fold).
Similarly, for ATF3 in the absence of pX (Fig.
8), 1 µg of transfected
ATF3 expressor vector results in an approximately 3-fold repression (55); in the presence of pX, 1 µg of transfected ATF3 plasmid DNA brings about a 6-fold enhancement of
repression. The assay monitoring ATF3 repression is carried
out in the absence of forskolin stimulation; accordingly, pX expression
does not enhance the transcriptional efficacy of endogenous CREB, as
shown earlier in Fig. 7. Importantly, all the transfection assays
contained equal amounts of total expressor plasmid DNA, to account for
squelching. We observe at lower levels of transfected ATF3
expressor (Fig. 8) increased repression efficacy by ATF3
due to pX action.
We conclude that the viral X protein interacts directly in the hepatocyte with bZip transcription factors and effects increased DNA binding potential to their cognate DNA-binding site and, in doing so, brings about augmented transcription or repression efficacy.
In this study we employed in vitro and in
vivo (cellular) approaches to examine the interactions of pX and
several bZip transcription factors, especially those that play a role
in hepatocyte function. We employed the CREB/pX system (1) to establish
the in vitro conditions to detect direct and specific
protein-protein interactions (Figs. 1 and 2). We show (Fig. 3) that the
bZip proteins ATF3, ICER II, gadd153/Chop10, and NF-IL6
directly interact with pX. Furthermore, the mammalian two-hybrid assay
demonstrated direct interactions between pX and the bZip proteins
in vivo (Fig. 5), confirming our in vitro
observations. This is the first demonstration of direct interactions
between pX and bZip proteins in the cellular environment of the
hepatocyte.
The bZip proteins, which are shown to directly interact with pX, are
induced in response to environmental cues such as growth factors,
cellular stress, and cytokines. ATF3 and ICER II are induced as immediate early genes, in response to conditions of cellular
stress (55) and cAMP induction (45), respectively. ATF3
mRNA expression is of particular interest for pX interactions due
to its transcriptional induction in the hepatocyte following mechanical
or toxin-induced liver injury (55). Importantly, the ATF3
homodimer acts as a repressor of CRE-mediated transcription and forms
heterodimers with gadd153/Chop10 (55). Similarly, NF-IL6 is rapidly
induced in the liver during the acute phase response (42).
We demonstrate here the functional significance of these bZip/pX interactions by in vitro (Fig. 4) and in vivo assays (Figs. 5, 6, 7, 8). As we demonstrated previously with CREB (1), we observe that pX significantly enhances the DNA-binding potential of these bZip proteins, in vitro. Importantly, pX enhances the DNA binding potential of bZip proteins that are capable of DNA binding; it does not convert non-DNA-binding bZip proteins, such as gadd153/Chop10 or ATF3/gadd153/Chop10 heterodimer, to DNA binding.
In the hepatocyte, pX expression enhances the transcriptional efficacy of each of the bZip proteins tested. Employing the HIV-LTR as our model system, we show that the transcriptional activation by NF-IL6 in the hepatocyte is enhanced 3-fold by pX. This is in agreement with our earlier observations (1). Based upon the results of Figs. 3, 4, and 6, we conclude that 1) in vivo, NF-IL6 and pX interact directly, 2) these interactions increase the DNA binding affinity of NF-IL6 for its binding site, and 3) this increased affinity increases the transcriptional efficacy of NF-IL6 by pX.
Regarding the transactivating effect of pX on the HIV-LTR, it has been
demonstrated (18, 21, 75) that pX requires multiple cis-acting elements
(18) for full transactivation, including the sequences containing the
NF-IL6-binding site I at positions 158 to
178 of the HIV-LTR (18).
Although there is no direct evidence demonstrating HIV infection in
hepatocytes in vivo, HBV DNA has been detected in
CD4+ T lymphocytes and monocytes (76). Our results are
consistent with the possibility that the NF-IL6/pX interactions in T
lymphocytes lead to higher HIV expression, contributing to the observed
synergy between HIV and HBV infections (76). Recent studies have
explored the mechanisms of NF-IL6 activation of HIV-LTR in monocytic
U937 cells (77, 78). However, the effect of NF-IL6/pX interactions in
lymphocytic or monocytic cell culture systems is not yet
understood.
Importantly, pX also enhances the repression efficacy of bZip
repressors ATF3 and ICER II. We interpret the results of
the in vitro (Figs. 3 and 4) and in vivo (Figs. 7
and 8) assays to mean that direct interactions between pX and bZip
repressors, ICER II
and ATF3, increase their DNA binding
affinity, thus resulting in increased repression efficacy by pX.
This is the first report describing the involvement of pX in enhancing
transcriptional repression of genes responsive to bZip repressors
ATF3 and ICER II. The implication of this observation is
that pX, by targeting bZip transcription factors, can potentiate not
only specific transcriptional activation but also transcriptional repression of cellular genes. In light of the recent observation that
ATF3 is transcriptionally induced in injured liver (55), our observation is of importance and significance for hepatocyte physiology.
It is well established that CREB/ATF and possibly C/EBP proteins are required for the activation of transcription from the viral HBV enhancer I (79). The effect of the aforementioned inducible bZip proteins on the interaction of CREB with pX in the activation of HBV enhancer I-mediated transcription is unknown. Competition of the inducible bZip proteins with the constitutive CREB for interaction with pX may have direct implications in the progression of the viral infection. We are currently investigating the role of pX in the nucleus in mediating CREB/pX and/or bZip/pX interactions during hepatocyte growth and hepatocarcinogenesis.
In conclusion, the results presented in this study support the hypothesis that pX acts as an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing their transcriptional activation or repression of cellular and/or viral bZip responsive genes. Our results are the first to demonstrate the promiscuity of pX interactions with bZip proteins. Since the bZip proteins examined include both transcriptional activators and repressors, these findings open new areas for investigating HBV pathogenesis and may, in turn, provide new insights regarding the molecular mechanisms by which HBV alters cellular gene expression.
We thank Drs. M. Bina, N. Fausto, R. Gaynor, R. Maurer, C. Molina, R. Schneider, and V. Tesmer for providing us with requested plasmids and other reagents. We are especially grateful to Drs. S. K. Konieczny and S. Johnson for providing us with unpublished plasmids. We thank Drs. M. Bina, S. Broyles, and R. L. Hullinger for helpful discussions and critically reviewing the manuscript.