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INTRODUCTION |
The D-site binding protein
(DBP)1 is a member of the
basic/leucine zipper family (b/ZIP) and was first isolated by its
ability to bind and transactivate through an element in the serum
albumin promoter (1). DBP mRNA is detected in most tissues,
although the protein accumulates to high levels only in the nuclei of
adult liver, suggesting that it is posttranscriptionally regulated. This 43-kDa protein is also known to undergo a robust circadian rhythm,
with its expression highest at approximately 8 p.m. (2). This
characteristic leads to the identification of one of its most likely
target genes, the cholesterol 7
-hydroxylase gene (C7
H), which can
be transactivated by DBP and which also exhibits a significant diurnal
regulation (3). Other genes that appear to be regulated by DBP include
PEPCK (4), alcohol dehydrogenase (5), carbamyl phosphate synthetase
(6), and P450 CYP2C6 (7). DBP may also be
involved in the regulation of the clotting factor IX gene and recovery
of the Leyden phenotype form of hemophilia B (8, 9).
In addition to the b/ZIP domain, DBP also contains a distinct region
termed the proline- and acid-rich (PAR) domain, which consists of a
large number (16%) of proline, glutamate, and aspartate residues. This
unique region, found immediately amino-terminal to the basic domain,
shares 83% identity with two other b/ZIP transcriptional regulators,
thyrothropic embryonic factor (TEF) (10) and hepatic leukemia factor
(HLF) (11, 12), and together they form the subfamily of PAR proteins.
TEF is expressed in the rat anterior pituitary gland and is one of the
key factors in transactivating the thyroid-stimulating hormone
promoter (10). HLF was discovered through studies of the human pre-B
acute lymphoblastic leukemia bearing 17:19 chromosomal translocations
where the b/ZIP domain of HLF was found fused to the E2A
gene (11, 12). HLF expression has been detected in the liver, though
little is known about the roles of TEF and HLF in this tissue. All
three members of the PAR subfamily recognize related sequences and are
able to heterodimerize (10, 13, 14). Despite the highly conserved nature of the PAR domain, no unique function has been associated with
it. Mutational analysis of the TEF PAR domain identified a cluster of
six basic amino acids, referred to as the "basic extension," that
is thought to be important for DNA binding specificity (10).
In this study we have been able to define a role for the PAR domain of
DBP in the transactivation potential of this protein. Through the use
of deletion mutants within the context of the whole DBP protein, we
have demonstrated that sequences within the central 28 amino acids of
the PAR domain are essential for DBP to transactivate a site from the
C7
H promoter. Using GAL4 DNA binding domain fusion proteins, we have
shown that the PAR domain must act in conjunction with sequences within
the amino terminus of DBP to mediate transactivation. Interestingly,
cotransfection of full-length DBP or HLF was able to dramatically
increase the transactivation potential of these GAL4-DBP fusion
constructs. Cotransfection of an E1A vector was able to inhibit
DBP-mediated transactivation. Introduction of a p300 expression vector
was able to overcome the E1A-mediated inhibition. These results
indicate that DBP transactivation is a p300-dependent process.
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MATERIALS AND METHODS |
Nuclear Extracts and DNA Binding Assays--
Nuclear extracts
were prepared from Hep G2 cells transfected by calcium phosphate
coprecipitation with DBP and DBP/GAL4 expression vectors. After 48 h, three 100-mm tissue culture dishes transfected with each construct
were washed, and the cells were collected and spun at 1000 × g for 5 min. The pellet was resuspended in 4 ml of lysis
buffer (10 mM Hepes, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 3 mM MgCl2, 10% glycerol,
1 mM dithiothreitol, 1% Trasylol, and 1 µg/ml
leupeptin/pepstatin A) and ruptured with 10 strokes of a Dounce
homogenizer. The mixture was spun at 500 × g for 5 min, and the pellet was resuspended in 1 ml of the above lysis buffer.
Cells were again pelleted for 2 min at 15,000 × g in a microcentrifuge, and the protein was extracted by resuspension in 150 µl of lysis buffer with 0.27 M KCl. This mixture sat on ice for 15 min with intermittent mixing, was spun for 15 min at 15,000 × g, and the protein-containing supernatant was
stored at
70 °C.
Gel mobility assays were performed as described previously (8). The
sequences of the oligonucleotides used in these experiments are
as follows.
D-site 5'- TGGTATGATTTTGTAATGGGG ACTAAAACATTACCCCACCAT-5' C7
H 5' -CTTTGGATGTTATGTCAGCA CCTACAATACAGTCGTAAAG-5' 17-G4M 5'-AGCTTCGGAGGACTGTCCTCCG GCCTCCTGACAGGAGGCTTCGA-5' Oligonucleotides
1
3 for bandshift probes
Supershift experiments were carried out by the preincubation of
the antibody with the protein extracts in the bandshift binding reaction for 30 min on ice, followed by the addition of the
radiolabeled oligonucleotide. Rabbit antibodies raised against the
full-length recombinant DBP protein were used to detect DBP among
nuclear proteins and extracts in both bandshift and Western assays.
Expression and Reporter Constructs--
A DBP-responsive
reporter construct was created by oligomerization of the C7
H
oligonucleotide (see above) followed by blunting of the overhang using
Klenow polymerase. Size-selected material was then cloned into the
G-free/TATA/LUC construct at the SmaI site. This vector has
previously been described (9) and includes the luciferase gene cloned
downstream of the G-free cassette. This allows for both in
vitro transcription and mammalian transfection experiments to be
carried out with the same vector. A GAL4-responsive reporter construct
was created using the same G-free/TATA/LUC vector and involved the
recloning of the dimer GAL4 site from 17M2-G.CAT using
HindIII and PstI sites upstream of the TATA box.
All full-length DBP sequences were cloned into the pSCT expression
vector downstream of the cytomegalovirus promoter. The PARN construct
was created by insertion of an EcoRI/BamHI
fragment encoding the NH2-terminal portion of DBP (amino
acids 1-185) upstream of the EcoRI/HindIII
PAR-bZIP-COOH-terminal fragment (amino acids 188-325). Each PAR
deletion construct contains the NH2-terminal fragment with
the bZIP and COOH-terminal portions of DBP with decreasing amounts of
the PAR domain (PARN, amino acids 187-253; PAR2, amino acids 207-253;
PAR2.1, amino acids 214-253; PAR2.2, amino acids 226-253; PAR3,
amino acids 235-253; BXT, amino acids 243-253).
GAL4 fusion constructs were made using the GAL4 DNA binding domain
(amino acids 1-148) in the vector G4MpolyII. Vectors containing the
PAR domain alone were cloned into XhoI/KpnI,
downstream of the GAL4 DNA binding domain. Constructs, including the
NH2-terminal portion of DBP upstream of the PAR domain
deletions listed above, were also inserted using XhoI and
KpnI.
Cell Culture and Cotransfection Experiments--
Transient
transfection assays were carried out as described previously, using the
calcium phosphate precipitation method into Hep G2 cells grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with
10% fetal bovine serum. A total of 20 µg of DNA was transfected per
100-mm plate and consisted of 5 µg of the reporter construct and 3 µg of a
-galactosidase expression vector, pCH110 (Amersham
Pharmacia Biotech). In cotransfection experiments, 2 µg of pCMV-DBP
was added. Luciferase and
-galactosidase assays were carried out
according to the manufacturer's instructions (Promega). The constructs
used were either the multimerized C7
H DBP site in the
G-free/TATA/LUC construct or the GAL4 consensus sequence reporter
construct. In cotransfection experiments using the GAL4 fusions, 2 µg
of the-GAL4-DBP constructs were added, either alone or with 500 ng of
full-length DBP (CMV 28) and 5 µg of the reporter construct
17M2-GLUC. All transfections contained 3 µg of pCH110 (Amersham
Pharmacia Biotech) and expressed the
-galactosidase gene, which
acted as an internal control.
In cotransfection experiments with CBP (a gift of Dr. Richard Goodman)
and p300 and E1A (a gift of Dr. David Livingston) expression vectors, 1 µg of PARN was added, either alone or in combination with 0.25 µg
of E1A and differing concentrations of p300 or CBP (0.1-5 µg) and
2.5 µg of the reporter construct. All transfections contained 1.5 µg of the pCH110 control plasmid.
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RESULTS |
Deletion Analysis of the PAR Domain--
The three known members
of the PAR domain family, DBP, TEF, and HLF, share a domain of high
sequence identity but unknown function. To determine whether the PAR
domain (amino acids 188-253) was required for DBP function,
progressive internal deletions from amino acid 188 through the entire
PAR domain were created (see Fig. 1 for
PAR domain sequence and location of deletions). These constructs
preserved the amino-terminal half of DBP as well as the b/ZIP domain so
that changes in DNA binding specificity were expected to be minimal.
Previous studies have demonstrated the ability of DBP to transactivate
via the high affinity albumin D-site in Hep G2 cells. In this work we
have taken advantage of a reporter construct consisting of multimerized
DBP binding elements derived from the C7
H promoter (3) cloned in
front of the albumin TATA box and driving a G-free cassette as well as
the luciferase gene. This vector has proven to be more specific for DBP
than the albumin D-site element, as transactivation due to the presence of CCAAT/enhancer binding protein
in these cells is considerably reduced with this construct. Expression of the full-length DBP construct results in an over 250-fold increase in promoter activity (Fig. 1B) compared with the empty expression vector
(PSCT). Deletion of the first 20 amino acids
(PAR2) resulted in little change in transactivation.
However, progressive deletions through the next 27 amino acids resulted
in a steady loss of activity. The PAR3 construct was reduced to a
30-fold activation, whereas the BXT construct (which eliminates the PAR
domain completely) was essentially inactive. These results indicate
that the PAR domain is critical for DBP transactivation and that a core
region of close to 30 amino acids within this region is of particular
importance.

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Fig. 1.
Analysis of deletions through the DBP PAR
domain. A, the amino acid sequence of the DBP protein
from amino acids 188-306, comprising the PAR, basic, and part of the
leucine zipper domains, is illustrated. The location of various
deletions of the PAR domain are indicated by the horizontal
arrows, with the name of the corresponding mutant appearing
above the arrow. The sequences of the comparable
regions of TEF and HLF are also shown, with regions of sequence
identity with DBP being boxed. The shaded boxes
indicate the location of hydrophobic residues in the leucine zipper.
B, transactivation mediated by DBP bearing internal
deletions of the PAR domain. Mammalian expression vectors containing
DBP cDNAs with the internal deletions indicated above, were
introduced into Hep G2 cells using calcium phosphate transfection,
along with a luciferase reporter construct bearing repeated DBP binding
elements derived from the cholesterol 7 -hydroxylase gene promoter.
Transfection efficiencies were corrected using a -galactosidase
expression vector (pCH1101) and are expressed as a -fold increase in
activity over the reporter construct with the empty expression vector
(PSCT). PARN represents the full-length DBP
expression vector. The averages of three transfections are indicated
(solid boxes), with the standard deviations shown by the
shaded boxes. C, analysis of DBP proteins bearing
internal deletions of the PAR domain. Large scale transfections of Hep
G2 cells were carried out using the various DBP expression vectors, and
nuclear extracts were prepared using high salt extraction. These
nuclear extracts were tested for DBP DNA binding activity using a
bandshift assay with the cholesterol 7 -hydroxylase and albumin
D-sites. The names of the constructs are indicated above
each lane. PSCT refers to the transfection of the
empty expression vector, and MOCK refers to untransfected
cell extracts. The location of DNA-protein complexes is indicated
(B), as is the position of the free DNA
(F).
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To confirm that the decreased transactivation mediated by the PAR
domain deletions was not the result of changes in protein stability or
DNA binding, nuclear extracts of Hep G2 cells transfected with the
various constructs were prepared. Western blot analysis indicated that
all of the constructs produced comparable amounts of protein with the
expected mobility (data not shown). The extracts were then normalized
for equal amounts of DBP protein and were used in gel mobility shift
assays, with the DBP binding elements from the C7
H gene promoter
(
237 to
212) (3) and the D-site of the albumin promoter (1) as
probes. Two different probes were used to determine whether these
mutations altered binding specificity. All of the PAR deletions appear
to have a slight increase in DNA binding activity compared with the
wild type sequence. Little or no decrease in relative DNA binding
activity was observed with either probe until a region known as the
basic extension was removed (Fig. 1C, BXT), which
resulted in complete loss of DNA binding activity. This is consistent
with the reported involvement of this region in modulating DNA binding
specificity in TEF. In contrast to the other deletion mutants, the
DNA-protein complex formed with the PAR3 construct was
characteristically diffuse. This change in migration may be associated
with conformational changes in DBP or alterations in binding of
associated proteins. Overall, these results indicate that the observed
decrease in DBP-mediated transactivation upon removal of parts of the
PAR domain (through to PAR3), is the result of changes in intrinsic transactivation activity and not the result of reduced DNA binding activity. Clearly, removal of the basic extension does have a dramatic
effect on DNA binding, resulting in complete loss of transactivation.
Transactivation mediated by DBP has previously been shown to be
tissue-specific, being active in Hep G2 cells, which are derived from a
hepatocellular carcinoma but inactive in L cells (1). To determine
whether the PAR domain plays a role in this tissue-specific activation,
the cotransfection experiments with the PAR deletions were preformed in
CV-1 cells, a monkey kidney cell line. No transactivation was observed
in this cell line even with the deletions (data not shown), indicating
that the PAR domain does not play a negative role in the generation of
tissue specificity. As DBP only transactivates in Hep G2 cells, it is
possible that other factors that are liver-specific may be required for
DBP transactivation.
The PAR Domain Is Part of the Transactivation Domain--
To
determine whether the PAR domain functions independently as a
transactivation domain, fusion constructs with the GAL4 DNA binding
domain were created. Each of the PAR deletion constructs, in
conjunction with the entire NH2-terminal portion of DBP
(amino acids 1-185), was fused upstream of a 147-amino acid region of GAL4, encoding its DNA binding domain in an SV40-based expression vector. The GAL4-responsive reporter construct consisted of two repeats
of the GAL4 binding site cloned into the G-free/TATA/LUC reporter
construct (see Fig. 3A). Transfection with a construct containing the entire amino-terminal and PAR domains of DBP into Hep G2
resulted in a 24-fold increase in activity (Fig.
2A, GNPN) compared
with transfection of the GAL4 DNA binding domain alone (G4M).
Progressive deletion of the PAR domain in the presence of the amino
terminus resulted in a decrease in transcriptional activity similar to
that seen with the whole protein (Fig. 2A: GNP2,
11-fold; GNP2.1, 11-fold; GNP2.2, 4-fold; and
GNP3, 4-fold). This effect of the PAR domain was dependent
on sequences within the amino terminus, as the PAR domain alone
(GP, 0.9-fold) exhibits no activity.

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Fig. 2.
Transactivation and DNA binding activity
mediated by GAL4 fusion proteins containing DBP amino and PAR
domains. A, the DBP amino-terminal region, in
conjunction with various segments of the PAR domains (as indicated in
Fig. 1), was inserted downstream of the GAL4 DNA binding domain in a
mammalian expression vector. GP is the PAR domain without
the amino-terminal region of DBP. These constructs were then assayed
for transactivation of a GAL4 reporter construct in Hep G2 cells.
Transfection efficiencies were corrected using a -galactosidase
expression vector and are expressed as a -fold increase in activity
over the reporter construct with the GAL4 expression vector. The
averages of three transfections are indicated (solid boxes),
with the standard deviations shown by the shaded boxes.
B, bandshift assays using a GAL4 recognition site were
carried out with extracts prepared with the GAL4 expression constructs
(left). The location of DNA/protein complexes is indicated
(B), as is the position of the free DNA (F). A
DBP polyclonal antibody was also added to the bandshift reaction
(right, +Ab). The location of the supershifted
complexes is indicated (SS), with the typical DBP complex
remaining in the well. The construct containing the PAR domain alone
produced a distinct supershifted complex.
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To address whether the deletions alter protein stability or DNA binding
activity, nuclear extracts from Hep G2 cells transfected with each of
the GAL4-DBP deletion constructs were prepared. Western blot analysis
using a DBP antibody indicated that all of the fusion proteins produced
comparable amounts of protein (data not shown). Bandshift assays with
these extracts were also carried out using the GAL4 recognition site.
The complexes formed with the GAL4 fusion containing only the PAR
domain (Fig. 2B, GP) or the GAL4 DNA binding
domain alone (G4M) were distinct. However, the
other N-terminal-containing proteins formed relatively diffuse
complexes. A supershift experiment with a DBP antibody was used to
specifically identify the fusion protein binding component (Fig.
2B, +Ab). All of the DBP-containing proteins
produced a comparable amount of supershifted complex (SS)
(slightly less GNP2.2 was added in this experiment). Overall, these
results suggest that the differences in transactivation mediated by the
GAL4 fusions are likely because of an intrinsic activity of these
proteins rather than changes in the amount or DNA binding activity of
the proteins produced.
The PAR Proteins Modulate DBP Transactivation--
Deletions of
the PAR domain in the context of the GAL4 constructs produced an effect
very similar to that observed with the whole protein. However,
comparison of the level of transactivation obtained with the GAL4
fusions with that of the whole DBP protein or with GAL4 constructs
containing the VP16 activation domain revealed that the activity of the
GAL4-DBP fusion construct was relatively low. The only domain not
included in the GAL4 constructs was the b/ZIP domain, which is involved
in DNA binding and dimerization. This disparity in activity suggests
that either dimerization was required for optimal transactivation or
that the presence of a functional protein increased transactivation.
The GAL4 transactivation experiments were repeated in the presence of
expression vectors for full-length DBP or the related PAR protein, HLF.
DBP and HLF together produced the most dramatic increase in
transactivation, with over a 10-fold increase in activity (Fig.
3B). This activity was
dependent on the presence of both the amino-terminal domain and the PAR
domain, as constructs containing either domain alone (Fig.
3A, GPN and GNP3) exhibited little
stimulation in the presence of the PAR proteins (Fig. 3B).
Interestingly, CCAAT/enhancer binding protein
, which recognizes
many of the same sites as DBP, was unable to activate the GAL4-DBP
fusion construct (data not shown). The absence of the b/ZIP domain in
the GAL4 fusion proteins should prevent interaction between the fusion
protein and the full-length PAR proteins. Attempts to demonstrate
physical interaction, either in vivo or in vitro,
have failed. These results suggest that the full-length PAR proteins in
some way modulate the activity of cellular components involved in
transcriptional activation. The requirement of this modulated factor
for optimal transactivation mediated by DBP suggests that this factor
has the properties of a coactivator.

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Fig. 3.
Cotransfection of DBP and HLF augments
transactivation by the GAL4-DBP fusion proteins. A, a
schematic diagram of the GAL4 constructs with the amino and PAR domains
(GNPN), the PAR domain alone (GPN), the amino
terminus and the PAR3 deletion (GNP3), or the vector
containing the GAL4 DNA binding domain alone
(G4M) is shown. The reporter construct is also
pictured, consisting of two repeats of the GAL4 recognition element
(2X GAL4 R.E.), a TATA sequence derived from the albumin
gene (ALBUMIN TATA), a G-free cassette (G-FREE),
and the luciferase gene (LUCIFERASE). B, the
GAL4-DBP fusion constructs were transfected into Hep G2 cells either
with the reporter construct or with expression vectors for both wild
type full-length DBP and HLF genes (+DBP/HLF). Transfection
efficiencies were corrected using a -galactosidase expression vector
and are expressed as a -fold increase in activity over the reporter
construct with the GAL4 expression vector (G4M).
The averages of three transfections are indicated (solid
boxes) with the standard deviation shown by the shaded
boxes.
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DBP Transactivation Is a p300-dependent
Process--
The adenoviral early protein E1A has been shown to
inhibit specific gene expression, as well as the induction of
differentiation, in several model systems by inhibiting the
coactivators p300 and CBP (15). To determine whether these proteins
play a role in DBP-mediated transactivation, a 12 S E1A expression
vector was cotransfected along with the wild type DBP vector and the
multimerized C7
H-site reporter construct. Binding of DBP to its
response element in this experiment resulted in a 250-fold increase in
activity (Fig. 4A). The
coexpression of E1A reduced this activity by a factor of 10, suggesting
that a protein bound and inactivated by E1A was involved in this
process. To directly test whether it is p300 or CBP that is required
for DBP activity, complementation of the E1A inhibition was attempted
using expression vectors for both proteins. Increasing concentrations
of a p300 expression vector were introduced along with the
E1A-containing vector. Relatively low concentrations of the p300
expression vector were able to completely restore DBP transactivation
(Fig. 4A), though this effect decreased at higher p300
levels. The effect of introducing p300 alone, without E1A, was also
examined. At the highest concentration of p300 vector used,
DBP-mediated transactivation was reduced to approximately 10% (Fig.
4A), the same level as observed with the E1A expression
vector. The tendency of p300 to form inactive aggregates when
cotransfected may sequester the endogenous p300, as has been observed
previously (16), resulting in the reduced activity observed. In the
presence of E1A, overall p300 levels must be stabilized, allowing for
complementation. Cotransfection of an expression vector for CBP, which
can also bind E1A, did not have any effect on DBP transactivation or on
the inhibition of transactivation by E1A (Fig. 4B). Even
though both p300 and CBP bind E1A, it is possible that p300 simply
sequesters E1A, preventing it from inhibiting some other cellular
factor. A p300 mutant that lacks the E1A binding domain
(p300
30) but is still functional (16) was used in the
same complementation assay. It resulted in comparable complementation
to the wild type p300 construct (Fig. 4C). Interestingly,
expression of the p300
30 construct alone did not inhibit
transactivation. This may be the result of a reduced tendency of this
p300 mutant to aggregate (16). Overall, these experiments demonstrate
that DBP transactivation requires a coactivator that can be inhibited
by the adenoviral E1A protein and that this coactivator is likely
p300.

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Fig. 4.
p300 overcomes the E1A-mediated inhibition of
DBP transactivation. A, a DBP expression vector
(PARN) was cotransfected into Hep G2 cells along with the
C7 H reporter construct. A 12 S E1A expression vector was also added
in conjunction with the other plasmids (+E1A). In addition,
the indicated amount (in µg) of a p300 expression vector was also
included with (PARN+E1A+p300) or without
(PARN+p300) the E1A vector. The -fold increase in activity
in relation to the reporter and empty expression vector
(PSCT) is indicated. The averages of three transfections are
indicated (solid boxes) with the standard deviations shown
by the shaded boxes. B, as above, except with the
addition of an expression vector for CBP, with
(PARN+E1A+CBP) or without (PARN+CBP) E1A.
C, as above, except with the addition of an expression
vector for p300 30, which contains a deletion that removes the E1A
binding region of p300 but otherwise maintains its activity.
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DISCUSSION |
The members of the PAR domain family of proteins are unique among
the b/ZIP class of factors in having an extended region of homology
outside of the b/ZIP domain itself. Previous studies have implicated a
relatively restricted part of the PAR domain, called the basic
extension, in site-specific recognition (10). However, a role for the
relatively large remaining portion of this domain has not previously
been defined. We have been able to demonstrate a requirement for this
domain in transactivation mediated by DBP. Deletions that remove
amino-terminal parts of the PAR domain but that do not effect DNA
binding activity are severely debilitated in their ability to activate
transcription via a high affinity DBP binding site. These same
sequences are required for activity of the GAL4 fusion proteins, and
again this region functions in conjunction with other sequences present
in the amino-terminal part of DBP, as either domain alone is inactive. The amino-terminal domains of all three PAR proteins have a 28-amino acid region of high similarity that may comprise the functional part of
the amino-terminal region of DBP (17). It has been shown that this
isolated segment from TEF acts as a transcriptional activator when
assayed outside the context of the whole protein (10). We have found
similar results with this amino-terminal region of DBP (data not
shown). It may be that other sequences in these proteins inhibit the
amino-terminal activation domain, which the PAR domain overcomes.
Alternatively, the isolated amino-terminal activation domain may
interact with a different part of the transcriptional machinery
compared with the intact protein. The amino-terminal and PAR domains
may represent separate contact points for the same protein or may
contact two different proteins where both are required for function.
The conserved nature of both the amino-terminal and PAR domains of all
three known PAR domain proteins, coupled with their similar tissue
distribution, suggests that they interact with the same protein or a
closely related factor.
Transcriptional activation occurs either through the direct
recruitment of parts of the transcriptional machinery such as TFIID or
in conjunction with coactivators. The coactivator p300 is known to
interact with a wide variety of b/ZIP factors including CREB (18),
c-Jun (19), and CCAAT/enhancer binding protein
(20). It functions
as a histone acetyltransferase (21) and may act to create a chromatin
environment favorable for transcription (22). The p300 protein has been
implicated in the regulation of differentiation in a variety of systems
(23) and has been shown to undergo increased phosphorylation in
response to differentiation (24). The ability of p300 to overcome
E1A-mediated inhibition of DBP transactivation strongly suggests that
this coactivator is required for the transcriptional activity of DBP.
The inability of CBP to complement the E1A-mediated repression of DBP
clearly differentiates the functions of p300 and CBP in this context. The dependence of DBP transactivation on a coactivator, rather than
directly recruiting the transcriptional machinery, implies a
requirement for regulation of this activity. This may be linked to
changes in gene expression associated with terminal differentiation of
the liver when DBP is exclusively expressed (1). The ability of DBP and
HLF expression to increase DBP transactivation and to up-regulate the
DBP promoter (25) suggests that some form of autoregulation of PAR
protein activity involving p300 may occur in liver cells.