Repression of Hepatocyte Nuclear Factor 4
by Tumor Suppressor p53: Involvement of the Ligand-Binding Domain and Histone Deacetylase Activity
Yutaka Maeda,
Shawn D. Seidel,
Gang Wei,
Xuan Liu and
Frances M. Sladek
Environmental Toxicology Graduate Program (Y.M.), Departments of
Cell Biology and Neuroscience (S.D.S., F.M.S.) and Biochemistry (G.W.,
X.L.), University of California, Riverside, California 92521
Address all correspondence and requests for reprints to: Frances M. Sladek, Ph.D., Department of Cell Biology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, Riverside, California 92521.
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ABSTRACT
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Tumor suppressor p53 is known to inhibit transactivation by certain
nuclear receptors, and overexpressed p53 is known to correlate with
poor differentiation in liver cancer. Therefore, we investigated
whether wild-type p53 might also affect the function of hepatocyte
nuclear factor 4
1 (HNF4
1), an orphan receptor required for liver
differentiation. Our results show that HNF4
1-mediated
transactivation is repressed by p53 but that the mechanism of
repression is not due to inhibition of HNF4
1 DNA binding. Rather,
transfections with Gal4 fusion constructs indicate that the repression
is via the ligand-binding domain of HNF4
1. Furthermore, we found
that p53 in human embryonic kidney whole-cell extracts preferentially
bound the ligand-binding domain of HNF4
1 and that the
activation function 2 region was required for the binding. Competition
for coactivator CREB binding protein could not entirely account for the
repression but trichostatin A, an inhibitor of histone deacetylase
activity, could reverse p53-mediated repression of
HNF4
1. In contrast, p53-mediated repression of
transcriptional activation of the same promoter by another
transcriptional activator, CCAAT/enhancer-binding
protein-
, could not be reversed by the addition of
trichostatin A. These results suggest that p53, like other
transcriptional repressors, inhibits transcription by multiple
mechanisms, one of which involves interaction with the ligand-binding
domain and recruitment of histone deacetylase activity.
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INTRODUCTION
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p53 is a well characterized tumor
suppressor gene involved in cell cycle control, DNA repair, and
apoptosis. p53 carries out these diverse functions by binding DNA
response elements in the promoter regions of genes critical to those
cellular processes and activating those genes (e.g., p21,
GADD45, bax). p53 is also known to repress the transcription of other
genes that lack p53 response elements, including the c-fos,
c-jun, retinoblastoma, IL-6, and bcl-2 genes (reviewed in
Ref. 1). Whereas, by and large, the mechanism of
transcriptional repression by p53 is much less well understood than
that of transcriptional activation, several mechanisms have recently
been proposed: binding to TATA-binding protein (2);
squelching of transcription factors IIB and IID (3); and
sequestration of coactivator p300/CREB binding protein (CBP)
(4, 5). p53 has also been found to repress transcription
of genes containing p53-binding elements in their promoter regions by
recruitment of mSin3A and histone deacetylase (HDAC) activity (6, 7). Histone deacetylation is thought to keep chromatin in a
closed conformation, thereby inhibiting transcription (reviewed in Ref.
8).
There are several reports of p53 blocking transcription by
nuclear receptors. Nuclear receptors are a superfamily of
ligand-dependent transcription factors that play a critical role in
growth, development, and differentiation of nearly every cell type in
the body. Members of the superfamily include steroid receptors,
vitamins A and D receptors, TR, and a large number of orphan receptors,
some of which have recently been found to respond to fatty acids
(PPARs), bile acids (farnesoid X receptor), cholesterol derivatives
(liver X receptor), and xenobiotics (steroid and xenobiotic
receptor, benzoate X receptor, and pregnane X receptor)
(reviewed in Refs. 9, 10, 11, 12, 13). Like other
transcriptional activators, nuclear receptors are known to activate
transcription by binding specific DNA response elements in
promoter/enhancer regions of genes and subsequently recruiting a
variety of different coactivator complexes which, by some poorly
defined mechanism, activate transcription by RNA polymerase II
(reviewed in Ref. 14). p53 has been reported to block the
activation of several different nuclear receptors by binding the DNA
binding domain (DBD) of the receptor and, in at least some cases,
subsequently inhibiting DNA binding [TR (15), GR
(16), ER (17)].
Since overexpressed p53 is known to correlate with poor liver
differentiation in liver cancer (18, 19, 20), we wanted to
determine whether p53 might also affect hepatocyte nuclear factor 4
1
(HNF4
1), an orphan nuclear receptor (NR2A1) known to be important
for liver differentiation. In addition to liver, HNF4
1 is also
expressed in adult kidney, intestine, and pancreas but not
other tissues (reviewed in Ref. 21). HNF4
1 is
considered to be an orphan receptor in that a ligand has not yet been
definitively identified for it (22, 23). However, HNF4
1
is known to be essential for development in organisms ranging from
insects to mammals and to regulate many essential genes related to
nutrient transport and metabolism. For example, HNF4
1 positively
regulates genes involved in the transport of lipids and vitamins as
well as genes involved in lipid, amino acid, and glucose metabolism. It
also regulates genes involved in the regulation of several serum
proteins such as blood coagulation factors, erythropoietin, and
antithrombin III. HNF4
1 is directly linked to several human
diseases: the HNF4
gene has been found to be mutated in
maturity-onset diabetes of the young type 1, and HNF4
1 DNA binding
sites have been found to be mutated in patients with hemophilia B
Leyden and maturity onset diabetes of the young type 1
(21). Therefore, since a tissue-specific knockout
indicates that HNF4
1 is essential for proper functioning of the
adult liver (24), repression of HNF4
1-mediated
transcription is anticipated to impede the function of the liver and
possibly other organs as well.
Like other nuclear receptors, HNF4
1 recruits coactivators
of the p160 and p300/CBP families via an interaction that depends
primarily on the activation function 2 (AF-2) region (putative helix
12) in the ligand-binding domain (LBD) (25, 26, 27, 28, 29). These
coactivator complexes contain or are associated with histone acetylase
activity, which serves to decondense the chromatin, thereby
facilitating transcription initiation (8, 14, 30).
However, unlike most other receptors, HNF4
1 interacts with
coactivators and activates transcription in a constitutive fashion,
i.e. in the absence of an exogenously added ligand.
In the current work, using transient transfection analysis, we
show that p53 represses HNF4
1-mediated activation on a
HNF4
1-responsive promoter (derived from the apolipoprotein B (apoB)
gene) in several different cell lines. We also provide evidence
indicating that the repression is via a mechanism not previously
described for p53 but one that is reminiscent of corepressors. Namely,
repression occurs via interaction of p53 with the LBD of HNF4
1.
Furthermore, the interaction appears to be dependent on the presence of
the AF-2 and to result in recruitment of HDAC activity. Finally, we
present evidence suggesting that this mechanism of repression may not
be applicable to basic leucine zipper protein CCAAT/enhancer-binding
protein-
(C/EBP
), indicating that p53, like other corepressors,
inhibits transcription by multiple mechanisms.
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RESULTS
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p53 Represses HNF4
1-Mediated Transactivation in
Vivo
To determine whether p53 represses HNF4
1-mediated
transactivation, transient transfection assays were performed in three
different cell lines: Saos-2, a human osteosarcoma cell line that is
deficient in both endogenous p53 and endogenous HNF4
1 protein (our
unpublished results and this study); HepG2, a human hepatocellular
carcinoma cell line that is at least partially differentiated for the
liver phenotype and expresses endogenous HNF4
1 (Ref. 31
and our unpublished data) and low levels of wild-type p53 protein
(32); and 293T, a human embryonic kidney cell line that
transfects very well and displays no endogenous HNF4
1 protein but
contains high levels of wild-type p53 protein (see Fig. 1B
). The results indicate that in all
three cell lines, wild-type p53 repressed transactivation by
transfected HNF4
1 from a reporter construct containing four HNF4
1
sites from the human apoB promoter (Fig. 1A
, compare lanes 4, 8, and 11
to lanes 3, 7, and 10, respectively). In HepG2 cells, p53 also
repressed transcription of the reporter construct alone, presumably by
interference with the endogenous HNF4
1 protein (compare lane 6 to
lane 5). The repression was relatively greater in the Saos-2 cells than
in the HepG2 and 293T cells, which may or may not be related to the
fact that the Saos-2 cells do not express endogenous p53 protein. To
verify that repression was not due to a loss of expression of HNF4
1
protein, immunoblot analysis was performed on the same whole-cell
extracts from 293T cells as those used to determine the luciferase
activity in Fig. 1A
. The results indicate that the level of HNF4
1
protein was not affected by cotransfection of the p53 vector (Fig. 1B
, top panel; compare lane 3 to lane 2), indicating that the
repression is via blocking of HNF4
1 function.

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Figure 1. p53 Represses HNF4 1-Mediated Transactivation in
Several Different Cell Lines
A, Transient transfection assays were performed into the indicated cell
lines with the pMT7. HNF4 (HNF4 ) (+, 0.1 µg for Saos-2 and
HepG2 cells, 1.0 µg for 293T cells) and pcDNA-p53 (p53wt) (+, 0.1
µg for Saos-2 cells, 0.05 µg for HepG2 cells, and 1.0 µg for 293T
cells) expression vectors (-, appropriate amount of salmon sperm DNA
or pcDNA3.1 empty vector) and the ApoB.-85-47. E4.Luc reporter
construct (2 µg) (diagrammed at top, not drawn to
scale) as indicated and as described in Materials and
Methods. Shown is the mean luciferase activity in RLU from one
representative experiment of several experiments ± SD
of triplicate samples. Luc, Luciferase. B, Immunoblot analysis (IB) of
the same 293T whole-cell extracts used to determine the luciferase
activity in panel A with the indicated transfected vectors
(bottom) and the primary antisera (right)
as described in Materials and Methods. -HNF4 ,
445; -p53wt, DO-1. Material from approximately one tenth of a
well was loaded in each lane. Low transfection efficiencies of Saos-2
and HepG2 cells and endogenous HNF4 1 protein in HepG2 cells
prevented a similar experiment from being done in those cell lines.
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p53 Does Not Inhibit HNF4
1 DNA Binding
To determine whether p53 might repress HNF4
1-mediated
transactivation via inhibition of DNA binding, as has been reported for
other nuclear receptors (15, 16, 17), EMSA was performed with
a 32P-labeled oligonucleotide containing the
HNF4
1 element from the human apoB promoter and HNF4
1 and p53
proteins expressed in mammalian cells (Fig. 2A
). We used two different immunoaffinity
methods to purify p53: one was via an antisera (12CA5) that recognizes
the hemagglutinin (HA) tag on the N terminus, and the other was via an
antisera (421) that recognizes the C terminus of p53 (Fig. 2B
). (The
421 antisera has been shown previously to activate p53 DNA binding,
suggesting that it may change the conformation of the protein
(33).) The results indicate that, even in the presence of
excess amounts of p53 protein (up to 200 ng vs.
approximately 20 ng of HNF4
1 protein, as determined by immunoblot
analysis, data not shown), regardless of the p53 purification method,
the ability of HNF4
1 to bind DNA was not altered (Fig. 2A
, compare
lanes 2, 3, and 4 to lane 1 and lanes 6, 7, and 8 to lane 5). This
suggests that the repression observed in vivo was not due to
an inhibition of HNF4
1 DNA binding but to an alternative
mechanism.
p53 Represses Activation by and Interacts with the HNF4
LBD
Since the HNF4
LBD is known to be essential for transactivation
in vivo, we hypothesized that p53 might interfere with the
function of the LBD. We therefore constructed a Gal4 fusion with the
HNF4
LBD (Gal4.HNF4.LBD) (Fig. 3A
) and
cotransfected it into 293T cells along with the p53 expression vector.
The results indicate that the Gal4.HNF4.LBD fusion protein activated
transcription well (Fig. 3B
, compare lane 4 to lane 3), as expected
from previous results (34). However, cotransfection with
the p53 expression vector repressed the Gal4.HNF4.LBD-mediated
transcription to approximately 50% (compare lane 6 to lane 4) but had
no significant effect on the basal activation by the Gal4 DBD (compare
lane 5 to lane 3), which, in any case, was only slightly above the
reporter alone (compare lane 3 to lane 1). Similar p53-mediated
repression of activation by Gal4.HNF4.LBD was observed in Saos-2,
HepG2, and 293 cells (data not shown). These results suggest that p53
inhibits transactivation by HNF4
by interfering with the function of
the LBD and are consistent with the notion that p53 does not inhibit
HNF4
1 DNA binding.
To determine whether p53 interacts with the HNF4
LBD, a
glutathione-S-transferase (GST) pull-down assay was
performed with whole-cell extracts from 293T cells, and the presence of
p53 protein was verified by immunoblot analysis. The results indicate
that the endogenous p53 protein bound very well (well over 10%) to the
GST.HNF4.LBD but only marginally to the GST.HNF4.DBD and not at all to
the GST control (Fig. 3C
, top panel; compare lane 4 to lanes
3 and 2). Since 293T cells express the SV40 large T antigen, and since
large T antigen is known to interact with p53 (33), we
performed a similar experiment with whole-cell extracts from 293 cells
that lack the large T antigen and observed the same result, namely that
the endogenous p53 protein bound the HNF4.LBD but not the GST control
or the HNF4.DBD (Fig. 3C
, bottom panel; compare lane 4 to
lanes 2 and 3). Since p53 also repressed transactivation by
Gal4.HNF4.LBD in 293 cells (data not shown), these results suggest that
repression of and interaction with the HNF4
LBD by p53 is not
mediated via the SV40 large T antigen.
Since the AF-2 region of the LBD is essential for interaction of
nuclear receptors with coactivators and corepressors (14),
we next determined whether the AF-2 of HNF4
1 was also required for
interaction with p53. The results indicate that the LBD construct
lacking the AF-2 (GST.HNF4.127.360) (see Fig. 3A
) did not bind p53
protein in 293T whole-cell extracts (Fig. 3D
, compare lane 4 to lane
5), indicating that the AF-2 is required for interaction with p53 and
suggesting that inhibition is via the AF-2 of HNF4
1. (The construct
lacking the AF-2 did, however, bind in vitro translated
HNF4
1, indicating that the lack of binding to p53 was not due to a
gross malfolding of the bacterially expressed GST protein; data not
shown).
Repression by p53 Is Only Partially Recovered by Overexpression of
CBP
The AF-2 of nuclear receptors is known to interact with
coactivators CBP/p300 (reviewed in Ref. 35), and p53 is
known to interact with p300 (36) and repress activation by
other transcriptional activators via competition for p300 (4, 5). Therefore, to determine whether repression of
HNF4
1-mediated transactivation is due to sequestration of limiting
amounts of an endogenous coactivator such as CBP/p300, we overexpressed
CBP in the transient transfection assay. The results indicate that CBP
enhanced the ability of HNF4
1 to activate its reporter construct
both in the absence and presence of cotransfected p53 (Fig. 4
, compare lane 6 to lane 4 and lane 7 to
lane 5). However, they also indicate that even in the presence of the
exogenously added CBP, the cotransfected p53 still reduced the
luciferase activity to approximately 60% compared with 46% in the
absence of added CBP (lanes 7 and 5). These results indicate that there
is only limited reversal of the p53-mediated repression by
cotransfection with CBP, indicating that a mechanism of repression
other than sequestration of CBP by p53 is involved.

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Figure 4. p53 Repression Is Only Partially Recovered by
Overexpression of CBP
Transient transfection assay in 293T cells with the ApoB.-85-47 Luc
reporter construct (2 µg) and the indicated expression vectors and 5
µg of coactivator CBP (pRC/RSV-mCBP.HA.RK) as described in Fig. 1A .
Percentages show RLU relative to samples lacking the p53wt vector.
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p53-Mediated Repression of HNF4
1 Is Reversed by HDAC Inhibitor
Trichostatin A (TSA)
The AF-2 of nuclear receptors has also been found to be involved
in the interaction of corepressors that recruit HDAC activity to
promoters (14). Since p53 has been shown to directly
repress transcription of target genes by recruiting HDAC1 to a promoter
containing a p53 binding site (6, 7), we reasoned that p53
might also repress HNF4
1-mediated transcription by recruiting HDAC
activity. To test this hypothesis, transient transfection assays into
293T cells with the Gal4 fusion constructs were performed in the
presence of TSA, a specific inhibitor of HDAC activity (37, 38). The results indicate that in the presence of TSA, the
repression by p53 of the Gal4.HNF4.LBD-mediated transactivation was
completely reversed (Fig. 5A
; compare
lane 8 to lane 6 vs. lane 4 to lane 2), indicating that
recruitment of HDAC activity to the HNF4
LBD may be involved in
p53-mediated repression.
We next determined whether TSA could also reverse the p53-mediated
repression of full-length HNF4
1 by using the HNF4
1 reporter
construct ApoB.-85 -47.E4.Luc. The results indicate that TSA
partially reversed the repression of HNF4
1 by p53 (Fig. 5B
; compare
lane 4 to lane 2). The significance, if any, of the fact that 100%
reversal was achieved with the Gal4.HNF4.LBD, but not the full-length
HNF4
1, is not known but could be due to the fact that other domains,
such as the DBD, the AF-1, and the F domain, are present in the
full-length HNF4
1 construct but not the Gal4.HNF4.LBD construct.
Since we had previously reported that the -85 to -47 region of
the apoB promoter also responds to the basic leucine zipper protein
C/EBP
(39), and since C/EBP
has been shown
previously to be repressed by p53 (40), we tested whether
that repression might also be reversed by TSA. The results indicate
that treatment with TSA did not reverse the repression of C/EBP
by
p53 (Fig. 5B
; compare lane 8 to lane 6), suggesting that repression of
C/EBP
by p53 may act via a mechanism other than recruitment of HDAC
activity. These and the previous results suggest that the effect of the
reversal of p53-mediated repression by TSA is a specific one and might
be related to interaction with the LBD.
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DISCUSSION
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In the current study we show for the first time that tumor
suppressor protein p53 can repress the constitutive transactivation
activity of orphan receptor HNF4
1 in vivo (Fig. 1
). We
also show that the repression is not due to an inhibition of HNF4
1
DNA binding by p53 but rather to an inhibition of LBD function (Figs. 2
and 3
). We show that endogenous p53 in 293T and 293 whole-cell extracts
can interact in vitro with the HNF4
LBD in an
AF-2-dependent fashion (Fig. 3
). Repression could not be completely
accounted for by competition for limiting amounts of coactivators such
as CBP (Fig. 4
). Rather, it appeared to be due, at least partially, to
recruitment of HDAC activity, as it was reversed by addition of TSA, a
specific inhibitor of HDAC activity (Fig. 5
). In contrast, repression
of basic leucine zipper protein C/EBP
by p53 could not be reversed
by TSA (Fig. 5
), suggesting that there may be multiple mechanisms of
repression. Taken together, our results support the notion that p53
represses transactivation of at least certain nuclear receptors by a
mechanism other than inhibition of DNA binding and one that involves
the LBD, the AF-2, and recruitment of HDAC activity.
New Mechanism of p53-Mediated Repression
Previous work by others showed that p53 inhibited transactivation
of certain nuclear receptors by interaction with the DBD and inhibition
of DNA binding (15, 16, 17, 41). Our results indicate that p53
also inhibits transactivation by HNF4
1 albeit via a different
mechanism, one that is more reminiscent of a corepressor than an
inhibitor of DNA binding. Hallmarks of corepressors include repression
of nuclear receptors only in the absence of ligand, interaction with
the receptor LBD, and recruitment of HDAC activity (14),
conditions all found in this study. What is not known at this point is
whether binding of the endogenous p53 in the cell extracts to the
HNF4
LBD is direct or indirect. Whereas we ruled out involvement of
the SV40 T antigen (Fig. 3
), it is still possible that some cellular
protein or another viral protein is required for the interaction.
However, it should be noted that p53 repressed activation by HNF4
1
in cell lines lacking known viral genes (Fig. 1
; Saos-2 and HepG2) and
that repression was via the LBD in those cell lines as well (data not
shown). Finally, it appears that repression via interaction with the
LBD may be applicable to other nuclear receptors as we have evidence
that repression of RAR/RXR activity by p53 is reversed by the addition
of ligand, suggesting involvement of the LBD (data not shown).
Multiple Mechanisms of p53-Mediated Repression
In addition to inhibition of DNA binding and recruitment of HDAC
activity, there appears to be additional mechanisms of repression
mediated by p53 since TSA did not completely reverse the repression of
full-length HNF4
1 (Fig. 5B
). One such mechanism could be competition
for coactivators such as CBP (Fig. 4
). p53 also repressed C/EBP
by a
mechanism other than recruitment of HDAC activity (Fig. 5B
), which may
or may not involve inhibition of C/EBP
DNA binding (40, 42). Others have also found multiple mechanisms of repression by
p53 of another liver-specific gene, although both of those mechanisms
required binding of p53 to a p53 response element (43).
This mechanism, however, is apparently not involved here as we saw no
evidence of p53 binding the ApoB element in the gel shift assay (Fig. 2A
), and p53 did not repress the ApoB reporter construct in the absence
of HNF4
1 in Saos-2 cells (Fig. 1A). The existence of
multiple mechanisms of repression is evidently not uncommon for
corepressors. Two well characterized transcriptional repressors,
silencing mediator of retinoid and thyroid hormone receptors (SMRT) and
retinoblastoma protein (Rb), have also been found to repress
transcription by multiple mechanisms, one of which involves recruitment
of HDAC activity under certain conditions and another that involves
interaction with the basal transcription machinery
(44, 45, 46).
Consequences of p53-Mediated Repression
HNF4
and C/EBP
are both critical for liver function and
differentiation (24, 47, 48). Therefore, repression of
their transcriptional activity by p53 could explain why overexpression
of p53 has been shown to correlate with poor liver differentiation in
liver cancer (18, 19, 20). Furthermore, p53-mediated
repression of C/EBP
and HNF4
1 could play a role in liver toxicity
of adenoviral vectors, such as those used in gene therapy, as an
increase in p53 protein levels due to adenovirus infection correlated
with a decrease in albumin gene expression, presumably via inhibition
of C/EBP
/ß transcriptional activity (40). Under
normal conditions in vivo, however, wild-type p53 protein
levels are very low and are increased only in response to cellular
stress such as DNA damage, withdrawal of growth factors, and hypoxia
(reviewed in Ref. 49). It has been assumed, with good
reason, that tight regulation of p53 protein levels is required to
prevent blockage of the cell cycle and induction of apoptosis. Perhaps
another reason to keep p53 protein levels low is to avoid interference
with normal cellular processes such as differentiation. Finally, an
increase in p53 protein levels may also serve as a mechanism to prevent
commitment of cellular resources for processes such as differentiation
during times of stress.
In conclusion, this report of p53-mediated repression of an additional
nuclear receptor suggests that, in addition to cell cycle control, DNA
repair, and apoptosis, repression of transcriptional activators
involved in cellular differentiation might be a general property of
p53. And, like those other processes, it appears that there are
multiple mechanisms by which p53 carries out this repression, one of
which is to interact with the LBD and recruit HDAC activity.
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MATERIALS AND METHODS
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Plasmids
The expression vectors containing rat HNF4
1
(pMT7.HNF4
1) (50), wild-type human p53 in pcDNA1
(pcDNA-p53) (51), and rat basic-leucine zipper protein
C/EBP
(pMT2.C/EBP
) (52) have been described
previously. The HNF4
GST fusion constructs were made by inserting
PCR products from the original rat HNF4
1 cDNA (53)
corresponding to fragments containing amino acids (aa) 127374
(GST.HNF4.LBD), aa 45125 (GST.HNF4.DBD), and aa 127360
(GST.HNF4.127.360) into the EcoRI/XhoI sites of
the pGEX6P-1 vector (Pharmacia Biotech, Uppsala, Sweden),
using appropriate primers containing EcoRI (sense) or
XhoI (antisense) sites. Gal4.HNF4.LBD (pFA-HNF4
.LBD) was
constructed by first inserting the appropriate
BamHI/NotI fragment from the GST.HNF4.LBD vector
containing aa 127374 of rat HNF4
1 into the pcDNA3.1 vector
(Invitrogen, Carlsbad, CA). Subsequently, the appropriate
BamHI/XbaI fragment from pcDNA3.1 was inserted in
frame with Gal4.DBD in the pFA-CMV vector (Stratagene, La
Jolla, CA). The reporter construct ApoB.-85-47.E4.Luc was constructed
by ligating four tandem copies of a double-stranded oligonucleotide
(ApoB.-85-47) corresponding to nucleotides -85 to -47
relative to the transcriptional start site of the human apoB gene and
containing a four-base overhang on the 5'-end (underlined)
(5'-GATCCGGGAGGCGCCCTTTGGACCTTTTGCAATCCTGGCGCTC-3',
top strand) into the BamHI site of pZLuc.E4, which contains
the adenovirus E4 TATA box driving the firefly luciferase gene.
pZLuc.E4 was generated by removal of an approximately 100-bp
BamHI fragment containing five RGC response elements from
5RGC.E4.Luc (54). The reporter construct pFR-luc contains
a synthetic promoter with five Gal4 binding elements driving the
expression of the luciferase gene (Stratagene). All PCR
constructs were verified by dideoxy sequencing. pRc/RSV-mCBP.HA.RK
containing full-length mouse CBP was kindly provided by R. Goodman.
Transient Transfection Assays
Cell lineshuman embryonic kidney cells (293 and 293T), human
hepatocarcinoma cells (HepG2), and human osteogenic sarcoma cells
(Saos-2)were routinely maintained in DMEM supplemented with
penicillin-streptomycin and 10% FBS (5% for 293 and 293T cells) at 37
C under 5% CO2. One day before transfection,
cells were seeded in six-well plates (Falcon 3046, Becton Dickinson and Co., Franklin Lakes, NJ) at 0.5 x
106 cells per well. DNA mixtures containing 2
µg luciferase reporter construct, 0.11.0 µg of HNF4
expression
vector, and 0.11.0 µg p53 expression vector (pcDNA-p53), as
indicated, were added to each well using Lipofectin according to the
manufacturers protocol (Life Technologies, Inc.,
Gaithersburg, MD). Total DNA was brought up to 10 µg with either the
appropriate amount of empty vector (e.g. pcDNA1 for samples
lacking p53) or salmon sperm DNA. After approximately 24 h,
transfected cells were harvested and luciferase activity was determined
as previously described (55). For cell treatment, cells
were treated with 100 ng/ml TSA (Sigma, St. Louis, MO) for
8 h before harvest. All assays were performed in triplicate in at
least two independent experiments, unless indicated otherwise. Results
are given in relative light units (RLU, an arbitrary unit) or fold
induction (RLU relative to transfections lacking expression vectors) as
indicated.
GST Pull-Down Assay
In vitro protein-protein interaction assays were
performed using GST fusion proteins expressed in E. coli
strain BL21(DE3)(pLysS) and bound to glutathione-agarose
(Sigma) using the manufacturers protocol
(Pharmacia Biotech). In general, 20 µl of a 1:1
GST-protein/glutathione agarose bead slurry (i.e. 10 µl
packed beads,
1020 µg protein) in 100 µl NETN
equilibration buffer (20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 1 mM EDTA, 0.01%
Nonidet P-40) were incubated with 100 µl of 10 mg/ml whole-cell
extract (see below) for 12 h at 4 C with gentle agitation. Protein
complexes were isolated by pelleting the beads at 3,000 rpm for 30 sec
in a Sorvall MC 12-V Microfuge (E.I. Dupont de Nemours & Co.,
Inc., Newtow, CT) and washing three times with 400 µl of
NETN equilibration buffer followed by resuspension in SDS-sample
loading buffer. After fractionation by 10% SDS-PAGE, proteins were
transferred to polyvinylidene difluoride membrane (Immobilon,
Millipore Corp., Bedford, MA) for immunoblot analysis (see
below). GST.HNF4.DBD beads were prewashed in buffer containing 1
M NaCl to remove contaminating DNA. Whole-cell
extracts were prepared by harvesting approximately
107 293 or 293T cells by gentle scraping in 1 ml
ice-cold PBS and resuspending in 0.2 ml ice-cold lysis buffer [50
mM Tris-HCl (pH.7.4), 150
mM NaCl, 5 mM EDTA, 0.5%
Nonidet P-40, 0.5 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol]. The lysis
mixture was incubated for 1 h at 4 C and then cleared by
centrifugation at 12,000 rpm for 15 min at 4 C.
Immunoblot Analysis
Immunoblot analysis was performed as previously described
(55) except that the blot was preincubated in blocking
buffer [5% nonfat dried milk, 0.01% sodium azide, 1x Tris-buffered
saline with Tween 20 (TBST)] and then incubated for 16 h
with a 1:5,000 dilution of
445 antiserum for HNF4
1
(53) or 0.5 µg/ml final concentration of DO-1 antibody
for p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
in blotting buffer (1% nonfat dried milk, 0.002% sodium azide, 1x
TBST). The blot was washed twice with 1x TBST and incubated for 2
h in the same blotting buffer with a 1:5,000 dilution of goat
antirabbit (for
445) or goat antimouse (for DO-1) IgG Fab'
conjugated to alkaline phosphatase (The Jackson Laboratory, West Grove, PA). The blot was washed three times in
1x TBST and developed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate toluidinium using a standard
protocol (56).
EMSA
Shift reactions and gel analysis were carried out as previously
described, with minor modifications (55). A standard
mobility shift reaction mixture (15 µl) contained 400 ng of COS-7
cell crude nuclear extract with overexpressed HNF4
1 incubated at 4 C
for 1 h with purified p53. The incubations were continued for
another 30 min at room temperature with 3 ng of
32P-labeled double-stranded oligonucleotide probe
(ApoB.-85-47) and nonspecific DNA [1 µg of poly(dIdC), 1 µg of
sonicated denatured salmon sperm DNA] before 5 µl were loaded onto a
6% native polyacrylamide gel in 0.25x Tris-borate-EDTA. After
electrophoresis, gels were subjected to autoradiography for 16 h
at room temperature. Crude nuclear extracts of pMT7.
HNF4
1-transfected COS-7 cells were prepared as previously described
(55). HA epitope-tagged wild-type human p53 was
immunoaffinity purified with either the 421 antibody (recognizes the C
terminus of p53) or the 12CA5 antibody (recognizes the HA tag at the N
terminus) antibody from HeLa cells infected with a recombinant vaccinia
virus as previously described (54, 57).
 |
ACKNOWLEDGMENTS
|
---|
We thank H. Brar, M. Mittal, J. Xun, S. Corneillie, S. Desai,
and L. Nepomuceno for technical assistance.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants DK-53892 to F.M.S. and CA-75180
to X.L. and fellowships to Y.M. from the Japan Society for the
Promotion of Science and University of California Toxic
Substances Research and Teaching Program.
Abbreviations: aa, Amino acid; AF-2, activation function 2;
apoB, apolipoprotein B; CBP, CREB binding protein; C/EBP
,
CCAAT/enhancer-binding protein-
; CREB, cAMP response element binding
protein; DBD, DNA-binding domain; GST,
glutathione-S-transferase; HA, hemagglutinin; HDAC,
histone deacetylase; HNF, hepatocyte nuclear factor; LBD,
ligand-binding domain; RLU, relative light units; TBST,
Tris-buffered saline with Tween 20; TSA, trichostatin A.
Received for publication June 1, 2001.
Accepted for publication October 1, 2001.
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