(Received for publication, November 1, 1995)
From the
Recently, Kalvakolanu et al. (Kalvakolanu, D. V. R.,
Liu, J., Hanson, R. W., Harter, M. L., and Sen, G. C.(1992) J.
Biol. Chem. 267, 2530-2536) showed that E1A inhibited the
basal and cAMP-stimulated transcription of the gene for
phosphoenolpyruvate carboxykinase (PEPCK). This inhibition was mediated
by the conserved region 1 (CR1) domain of E1A, which has been shown by
other laboratories to bind to the cellular transcriptional adaptor
proteins, p300 and cAMP response element binding protein (CREB)-binding
protein (CBP). The PEPCK gene promoter contains a functional
cAMP-response element, through which CREB and, therefore, CBP modulate
transcription, and a consensus p300 DNA binding sequence is also
present in a distal protein binding site of the promoter. We
hypothesized that E1A might inhibit PEPCK gene transcription by binding
to p300 and/or CBP. Surprisingly, we found that E1A consistently
stimulated basal transcription from the PEPCK promoter in transfection
assays in adenovirus (Ad)-infected HepG2 hepatoma cells or
E1A-expressing, stably transfected 3T3 fibroblasts and nuclear run-on
assays in Ad-infected H4IIE hepatoma cells. E1A also enhanced the
stimulation of PEPCK gene transcription by BtcAMP. In
transfection assays, wild type Ad5 expressing both 243R and 289R forms
of E1A or a mutant virus expressing the 289R form alone stimulated
transcription from the PEPCK promoter by approximately 5-fold 20 h
postinfection. However, no stimulation was observed in cells infected
with a virus expressing either the 243R protein alone or a 289R protein
from which conserved region 3 (CR3) was mutated. Mutation or deletion
of CR1 of E1A had no significant effect on transcription from the PEPCK
promoter. Mutations within conserved region 2 (CR2) of E1A that inhibit
the binding of E1A to the retinoblastoma gene product (pRb) further
enhanced the stimulation of transcription from the PEPCK promoter by
2-3-fold compared with wild type E1A. These findings suggested
that the normal function of pRb is to stimulate PEPCK gene
transcription, and that this process is inhibited by the binding of E1A
to pRb. This hypothesis was confirmed by overexpressing pRb in HepG2
cells, which stimulated transcription from the PEPCK promoter. Our
findings indicate that Ad E1A regulates PEPCK gene transcription
through a stimulatory mechanism involving CR3, and by attenuating a
stimulatory effect of pRb through CR2.
The nuclear phosphoproteins encoded by the adenovirus (Ad) ()E1A gene are essential for efficient Ad
replication(1) . The E1A gene consists of two exons
encoding 12 and 13 S mRNAs, which arise through alternative splicing of
a single pre-E1A RNA transcript(2) . In Ad serotypes 2 and 5
(Ad2 and Ad5) these transcripts translate 243R and 289R E1A proteins
that are identical except for a unique internal stretch of 46 amino
acids present in the larger protein. DNA sequence homology between Ads
of different groups is limited to conserved regions (CRs) located
entirely within the first exon of E1A(3) . Two CRs,
conserved regions 1 (CR1) and 2 (CR2), are present in both the 243R and
289R proteins, whereas the third domain, conserved region 3 (CR3), is
unique to E1A 289R.
Mutational analysis of the E1A gene has linked the CRs to many of the biological activities of E1A (i.e. transcriptional activation of both viral and cellular genes, enhancer-mediated transrepression, cellular transformation, etc.) (4) . Amino acids encoded within the CRs of E1A interact with specific cellular proteins, thereby transducing the numerous biological activities of E1A. For example, many of the transcriptional transactivation functions associated with E1A are mediated by CR3-encoded amino acids interacting with specific cellular transcription factors like TFIID(5, 6) , ATF2(6, 7, 8) , and activator protein 1 (AP-1)(9, 10) . On the other hand, CR1- and CR2-encoded amino acids serve as binding sites for the protein product of the retinoblastoma gene (pRb), thereby releasing the cellular transcription factor, E2F, in a transcriptionally active, non-pRb-bound form(11) . The enhancer-mediated transrepression function of E1A has been ascribed to both CR1 and CR2(4) .
The phosphoenolpyruvate carboxykinase (PEPCK) gene encodes the rate-determining enzyme in gluconeogenesis, and its transcription can be stimulated or repressed in response to a number of extracellular stimuli as well as by tissue-specific and developmental factors (12, 13, 14, 15) . Recently, Kalvakolanu et al. (16) demonstrated that E1A inhibited both basal and cAMP-stimulated transcription of the PEPCK gene. These data indicated that CR1-encoded amino acids, which are required for the interaction of E1A with the cellular nuclear proteins, p300 and CBP, were required for the inhibition of PEPCK gene transcription by E1A. A functional CRE that binds CREB, and therefore CBP, has been identified in the PEPCK gene promoter(12, 17) . Sequence analysis of the PEPCK gene promoter also shows that a region of the promoter (P6 ((12) ) or AF-1 ( (18) and (19) )) that plays important roles in glucocorticoid and retinoic acid responsiveness(18, 20) contains a consensus p300 binding sequence(21) . These preliminary findings led us to hypothesize that E1A could inhibit PEPCK gene transcription by interacting with p300 and/or CBP and blocking their transcriptional adaptor function. Alternately, E1A could inhibit PEPCK gene transcription by binding to p300, with subsequent binding of the E1A-p300 complex to the P6 region of the PEPCK gene promoter.
We report here that E1A, unexpectedly, stimulates basal PEPCK gene transcription even under conditions identical to those reported by Kalvakolanu et al.(16) . The stimulation of PEPCK gene transcription was mediated by CR3 of E1A. Transcription from the PEPCK promoter was further stimulated by a mutant E1A protein from which CR2 was deleted as well as with mutant E1A proteins containing point mutations in CR2 that abolish the interaction of E1A with pRb. Overexpression of pRb alone or in the presence of E1A stimulated transcription from the PEPCK gene promoter. These findings indicate that E1A regulates PEPCK gene transcription through two mechanisms. First, E1A stimulated transcription from the PEPCK promoter through CR3, presumably by interacting with a transcription factor(s) that binds to the PEPCK gene promoter. Secondly, E1A appears to attenuate a stimulatory effect of pRb on PEPCK gene transcription.
The eukaryotic expression
vector for wild type, human pRb, pRbWT-HA/SVE(29) was
provided by Robert Weinberg (Whitehead Institute for Biomedical
Research, Cambridge, MA). [-
P]UTP was
purchased from Dupont NEN. Vanadylribonucleosides, ATP, GTP, CTP, and
RNase-free DNase were purchased from Life Technologies, Inc. Luciferase
assay reagents were obtained from Analytical Luminescence Laboratory
(San Diego, CA), and chloramphenicol acetyltransferase (CAT)
enzyme-linked immunosorbent assay kits were from Boehringer Mannheim.
Plates of HepG2 or 3T3 cells were grown to approximately 80% confluency and transfected with the indicated plasmids by calcium phosphate-DNA coprecipitation as described by Park et al. (17) . Immediately following transfection, HepG2 cells were infected with the indicated Ads as described previously(31) .
Luciferase assays were performed on a
Monolight 2010 luminometer using the enhanced luciferase assay kit
(Analytical Luminescence Laboratory, San Diego, CA) according to the
supplier's directions. CAT assays were performed with CAT
enzyme-linked immunosorbent assay kits (Boehringer Mannheim) according
to the manufacturer's instructions. Transfection efficiencies
were normalized by cotransfecting the cells with a plasmid containing a
chimeric Rous sarcoma virus long terminal repeat -galactosidase
gene, and
-galactosidase levels were measured as described
previously(17) . All experiments were repeated three times, and
consistent results were obtained in all cases.
Figure 1: Ad E1A stimulates PEPCK gene transcription. HepG2 cells were transfected with a plasmid containing the full-length PEPCK gene promoter linked to the luciferase reporter gene (-490pPCLuc). The cells were then infected with the indicated Ads for 20 h. Luciferase activity in cell lysates was measured as described under ``Experimental Procedures.'' Levels of transcription are shown relative to transcription levels in mock-infected cells.
To confirm the observations using the PEPCK gene promoter-luciferase reporter gene in HepG2 cells, transcription of endogenous PEPCK gene was also measured by nuclear run-on transcription assays in mock or Ad5-infected H4IIE rat hepatoma cells. H4IIE cells were selected for nuclear run-on transcription assays since the endogenous PEPCK gene in HepG2 cells is poorly transcribed. Consistent with our observations using the PEPCK gene promoter-luciferase reporter gene in HepG2 cells, basal PEPCK gene transcription was stimulated approximately 20-fold in Ad5-infected-H4IIE (data not shown).
We have also found that Ad E1A
enhanced cAMP-stimulated transcription from the PEPCK promoter. When
uninfected HepG2 cells were treated with dibutyryl-cAMP
(BtcAMP), PEPCK promoter-driven transcription was
stimulated by approximately 5-fold (Fig. 2). However, treatment
of Ad5-infected cells with Bt
cAMP produced a
12-13-fold increase in transcription from the PEPCK promoter. Ad5
infection alone produced a 5-fold increase in transcription from the
PEPCK promoter as previously observed. No further increase in
Bt
cAMP-stimulated transcription was seen in cells infected
with dl343, which does not express E1A proteins (data not shown).
Figure 2:
Ad E1A enhances
BtcAMP-stimulated transcription from the PEPCK gene
promoter. HepG2 cells were transfected with -490pPCLuc and then
infected with wild type Ad5 or mock-infected for 20 h as shown. During
the last 4 h of infection the indicated cells were treated with 0.5
mM Bt
cAMP. Luciferase activity in cell lysates was
measured as described previously, and transcription levels are shown
relative to levels measured in mock-infected cells that were not
treated with Bt
cAMP.
The ability of Ad5 infection to stimulate transcription from series of luciferase reporter vectors containing the full-length PEPCK promoter in which individual protein binding sites had been mutated (12, 17, 34, 35, 36) was used to define E1A-responsive regions of the PEPCK promoter. We found that mutations in several protein binding sites including the 3` portion of the P3 element (P3(I), (12) ), which binds C/EBP proteins(17, 35, 36) , the P6 element, which has been shown to be involved in glucocorticoid and retinoic acid responsiveness(18, 20) , the P1 site(12) , which binds nuclear factor 1/CCAAT box transcription factor (NF-1/CTF), and C/EBP proteins(36) , or the distal cAMP-response element (CRE-2, (12) ), which also binds C/EBP proteins (36) , inhibited E1A-stimulated transcription by 30% and 75% (data not shown). Mutations in other protein binding sites (i.e. TATA box, CRE-1, P2, p3(II), P4, and P5) had no significant effect on E1A-stimulated transcription.
Figure 3: PEPCK gene transcription is stimulated by Ad E1A through CR3. A, HepG2 cells were transfected with -490pPCLuc, and then infected with the indicated Ads for 20 h. Luciferase activity was measured as described previously and transcription levels are shown relative to levels measured in mock-infected cells. B, 3T3 fibroblast stably expressing 12 or 13 S E1A proteins or containing an empty expression vector (None) were transfected with -490pPCLuc. After incubating the cells for 18 h, luciferase activity in cell lysates was measured as described previously. Levels of transcription are shown relative to levels measured in cells expressing no E1A protein (None).
To confirm the stimulation of PEPCK transcription by 289R E1A in the absence of viral infection, luciferase production from the PEPCK promoter was measured in MT 12-1 and 13-2, 3T3 cells that stably express either the 243R or 289R E1A proteins, respectively. Transcription from the PEPCK promoter was approximately 24-fold higher in 13-2 cells compared with control, 3T3-neo cells (Fig. 3B). No stimulation of transcription was observed in MT 12-1. These data indicate that E1A stimulates PEPCK gene transcription in a CR3-dependent manner.
Figure 4: CR1 ofE1A does not regulate PEPCK gene transcription. HepG2 cells were transfected with -490pPCLuc and then infected with the indicated Ads for 20 h. Luciferase activity in cell lysates was measured as described previously. Transcription levels are shown relative to levels measured in mock-infected cells.
Figure 5: Ad E1A regulates PEPCK gene transcription through CR2. HepG2 cells were transfected with -490pPCLuc and then infected with the indicated Ads for 20 h. Luciferase activity in cell lysates was measured as described previously. Transcription levels are shown relative to levels measured in mock-infected cells.
The previous mutations in CR2 abolish E1A binding to a number of cellular proteins (p107, p60 (cyclin A), pRb) (Table 1). However, the ``superstimulation'' of transcription from the PEPCK promoter was also observed in cells expressing mutant E1A proteins that are unable to bind pRb alone. The Ad mutant 13S-928, which contains a point mutation in CR2 specifically blocking E1A binding to pRb without affecting p107 or p60 binding stimulates transcription from the PEPCK promoter by 13-14-fold compared with the 5-fold increase with pm975 (Fig. 5). The lack of E1A-p300 binding in modulating PEPCK transcription is further illustrated by noting that RG2-928, which encodes E1A proteins (13 and 12 S) unable to bind both p300 and pRb does not increase the level of transcription from the PEPCK promoter more than cells infected with E1A-1108, which interacts with p300 normally. These data cannot be explained by the disparate levels of mutant E1A proteins in infected cells. Comparisons of matched sets of E1A mutants (E1A 13 S only expressing Ad: pm975, 13S928, DL2; E1A 12 and 13 S expressing Ad, Ad5, E1A-1104, E1A-1108, RG2-928) demonstrate equivalent levels of E1A proteins (data not shown) but consistently higher levels of PEPCK transcription with CR2 mutants that do not bind Rb.
Figure 6: pRb regulates PEPCK gene transcription. HepG2 cells were cotransfected with -490pPCLuc and increasing amounts of the pRb expression vector, pRbWT-HA/SVE. The numbers along the horizontal axis indicate the mol of pRbWT-HA/SVE to -490pPCLuc used in the transfections. The cells were then mock-infected or infected with wild type Ad5 as indicated for 20 h. Luciferase activity was measured in cells lysates as described under ``Experimental Procedures.'' Levels of transcription are shown relative to mock-infected cells not cotransfected with pRbWT-HA/SVE.
Previous studies by Kalvakolanu et al. (16) showed that the E1A proteins inhibited basal and cAMP-stimulated transcription of the PEPCK gene. This process was dependent upon CR1 of E1A, which has been shown to interact with the cellular transcriptional adaptors, p300 and CBP(37, 38, 39) . Since the PEPCK promoter contains a CREB/CBP binding site and a potential p300 consensus binding site, we hypothesized that the PEPCK promoter might be a valuable system for further analysis of E1A and p300/CBP transcriptional activity. Interestingly, rather than inhibiting PEPCK gene transcription, we have consistently observed that E1A stimulates basal and cAMP-stimulated transcription from the PEPCK gene promoter. Our findings also differ from those reported by Kalvakolanu et al.(16) in that only E1A 289R regulated transcription from the PEPCK promoter, while both 243R and 289R exhibited activity in the other study. In addition, we found that mutations in CR1 had little effect on PEPCK promoter-driven transcription, whereas both CR2 and CR3 influenced PEPCK gene transcription through independent mechanisms.
It is unclear why our findings differ so significantly from those reported by Kalvakolanu et al.(16) . The differences between our data and those reported by Kalvakolanu et al.(16) may be due to infection conditions or the PEPCK promoter-reporter gene plasmids used in each laboratory. For example, Kalvakolanu et al.(16) used MOIs that were significantly lower and times of infection that were much shorter than used in our studies. Under the infection conditions reported by Kalvakolanu et al.(16) we were unable to detect significant levels of E1A expression but observed small increases in PEPCK promoter-driven transcription (data not shown). We also found that E1A proteins stimulated transcription from the PEPCK promoter-CAT reporter gene plasmids (generously supplied by Richard Hanson, Case Western Reserve University, Cleveland, OH) used in the studies by Kalvakolanu et al.(16) . Therefore, we find that E1A stimulates transcription from the PEPCK gene promoter even under the conditions reported in the other study, and we are currently unable to explain the differences between our data and those reported by Kalvakolanu et al.(16) . However, we have consistently observed that E1A stimulates PEPCK gene transcription by multiple techniques and experimental systems. Transcription from the PEPCK promoter was stimulated by E1A 289R as measured by nuclear run-on transcription assays or by luciferase (and CAT) reporter gene analysis. Similar data were obtained in several different cell lines (H4IIE, HepG2, 3T3) and in Ad-infected cells or in stably transfected cells. Therefore, we have concluded, based on the findings of numerous experiments, that E1A 289R stimulates PEPCK gene transcription.
Our data indicate that E1A 289R regulates basal transcription from the PEPCK promoter by two mechanisms. The first mechanism involves the stimulation of transcription from the PEPCK promoter by CR3. This conclusion is based on the inability of both wild type 243R protein and a mutant 289R E1A protein from which CR3 was deleted to stimulate transcription from the promoter. This finding is consistent with the transcriptional transactivation function normally attributed to CR3 of E1A. For example, E1A has been shown to stimulate gene transcription by binding to ATF2(6, 7, 8) , TFIID(5, 6) , and the CCAAT box binding factor of the hsp70 promoter (40) through CR3. In other experiments, 289R has been shown to enhance the transcriptional activity of SP1(6) , implicating CR3 in this transcriptional processes.
A number of transcription factor binding sites have been identified
in the PEPCK promoter through which E1A could act. A TATA box, which
binds TFIID(12) , as well as a cAMP regulatory element (CRE-1),
which binds CREB/ATF and C/EBP proteins(17, 36) , and
a CCAAT/NF-1 site, which binds NF-1(12) , are present in the
PEPCK promoter. Sites that mediate glucocorticoid(18) , thyroid
hormone(41) , and insulin regulation (42, 43) have been defined, and other binding sites
for C/EBP proteins(17, 36) , hepatic nuclear factors
1, 3, 3
, and 4(12, 19, 44) , and
AP-1 (35) have also been identified. Although E1A has not been
shown to interact with all of these factors, there are potentially
numerous mechanisms by which E1A could regulate transcription from the
PEPCK promoter. This hypothesis is supported by the ability of several
mutations in individual protein sites in the PEPCK promoter to inhibit
the stimulation of transcription by E1A. Thus, E1A appears to stimulate
PEPCK gene transcription by interacting with multiple factors that bind
several sites in the promoter.
The second mechanism by which E1A
regulates PEPCK promoter-driven transcription is through an interaction
with pRb. The interaction between E1A and pRb appears to inhibit
transcription from the PEPCK promoter since transcription is
``superstimulated'' in cells expressing mutant E1A proteins
that are unable to bind pRb. This suggests that the normal function of
pRb is to stimulate PEPCK gene transcription. Although pRb is generally
characterized as a growth suppressor protein that inhibits gene
transcription, pRb has been shown to stimulate the transcription of
certain genes including the genes for TGF-1 (45) and the
fourth promoter of insulin-like growth factor II (46) . In
these instances pRb may stimulate transcription directly by binding to
certain transcription factors, or by inhibiting the expression or
binding of inhibitory transcription
factors(45, 46, 47, 48) .
A
simple model to account for the regulation of PEPCK gene transcription
by E1A is shown in Fig. 7. In uninfected cells, pRb stimulates
transcription (i.e. contributes to the normal basal level of
transcription) from the PEPCK promoter through an unknown mechanism (Fig. 7A). For simplicity we have shown pRb binding
directly to the promoter to indicate its stimulatory effect. In
Ad-infected cells, E1A stimulates transcription from the PEPCK promoter
by interacting with transcription factors through CR3 (for simplicity
this is also shown as E1A binding directly to the promoter) but
inhibits pRb-mediated stimulation by binding to pRb and preventing its
normal interactions (Fig. 7B). The
``superstimulation'' of transcription observed with mutant
E1A proteins that cannot bind pRb is due to stimulation of
transcription by E1A through CR3 and the ability of free pRb to enhance
transcription from the promoter (Fig. 7C). Finally, in
Ad-infected cells transfected with the pRb expression vector, E1A
stimulates transcription from the PEPCK promoter through CR3 but is
unable to bind and inactivate the increased levels of pRb that also
stimulate transcription, resulting in superstimulated levels (Fig. 7D). Thus, wild type E1A binds pRb and prevents
it from activating transcription. How pRb regulates PEPCK gene
transcription is unclear. Like the stimulation of transcription by E1A,
the mutation of several protein binding sites in the PEPCK promoter
inhibits the effect of pRb on transcription from the PEPCK promoter. ()Since pRb has not been shown to bind to the PEPCK
promoter, pRb may stimulate PEPCK gene transcription by inhibiting the
expression or activity of a transcription factor that inhibits PEPCK
gene transcription.
Figure 7: Model of Ad-stimulated PEPCK gene transcription. Wild type and mutant E1A proteins and pRb are indicated. The mechanism of stimulation by E1A and pRb is not known but is indicated by the direct binding of the proteins to the promoter. Levels of transcription are indicated by the thickness of the arrows.
Finally, we have shown that Ad E1A also enhances
cAMP-stimulated transcription from the PEPCK promoter. This data also
differs from the findings of Kalvakolanu et al.(16) who showed that E1A inhibited cAMP-stimulated PEPCK
gene transcription through CR1. Their findings are particularly
interesting since Arany et al.(38) and Lundblad et al.(39) recently reported that E1A also inhibits
cAMP-stimulated transcription from the somatostatin promoter and from a
Gal4-responsive promoter in the presence of a chimeric Gal4-CREB
protein. This inhibition was also mediated by CR1, which interacts with
CBP and p300. Presumably, E1A binds CBP and prevents it from
interacting with protein kinase A-phosphorylated CREB, thereby blocking
cAMP-stimulated transcription. In preliminary experiments, we have also
found that E1A blocks cAMP-stimulated transcription from the
somatostatin promoter and with the Gal4-CREB-responsive system.
However, E1A consistently enhanced BtcAMP-stimulated PEPCK
gene transcription in side-by-side assays.
Furthermore, in
preliminary experiments the dominant negative CREB inhibitor,
KCREB(49) , inhibited cAMP-stimulated transcription from the
PEPCK promoter but did not significantly block the enhanced levels of
transcription observed in the presence of both Bt
cAMP and
E1A. (
)However, mutation of the proximal cAMP-response
element (CRE-1), which binds CREB, completely blocked cAMP-stimulated
transcription and the enhanced levels of transcription observed with
E1A and Bt
cAMP. These findings suggest that E1A and cAMP
may regulate PEPCK gene transcription through a CREB-independent
mechanism. Studies by Hanson and colleagues (36) have shown
that the proximal CRE of the PEPCK promoter also interacts with C/EBP
proteins as well as AP-1. Their data support the hypothesis that C/EBP
or other proteins may also mediate the cAMP response of the PEPCK
promoter. In the future, we will address the ability of E1A to interact
with C/EBP proteins and AP-1 and the effect of these interactions on
cAMP-stimulated PEPCK gene transcription.